WO2017132330A1 - Compositions and methods for controlling insect pests - Google Patents

Compositions and methods for controlling insect pests Download PDF

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Publication number
WO2017132330A1
WO2017132330A1 PCT/US2017/015061 US2017015061W WO2017132330A1 WO 2017132330 A1 WO2017132330 A1 WO 2017132330A1 US 2017015061 W US2017015061 W US 2017015061W WO 2017132330 A1 WO2017132330 A1 WO 2017132330A1
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Prior art keywords
insect
plant
group
insecticidal
polynucleotide
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PCT/US2017/015061
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French (fr)
Inventor
Jodi Beattie
Michael John CRAWFORD
Brian Donovan EADS
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Monsanto Technology Llc
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Application filed by Monsanto Technology Llc filed Critical Monsanto Technology Llc
Priority to AU2017212500A priority Critical patent/AU2017212500B9/en
Priority to EP20170141.4A priority patent/EP3748003A1/en
Priority to RU2018130694A priority patent/RU2792660C2/en
Priority to CN201780014383.XA priority patent/CN109312344A/en
Priority to EP17744874.3A priority patent/EP3408390A4/en
Priority to US16/072,824 priority patent/US10975387B2/en
Priority to CA3011521A priority patent/CA3011521A1/en
Publication of WO2017132330A1 publication Critical patent/WO2017132330A1/en
Priority to US17/228,596 priority patent/US20210254094A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • Pest species of interest include flea beetles, such as Phyllotreta spp. and Psylliodes spp.
  • RNA interference RNA interference
  • invertebrate pests such as insects.
  • RNA interference RNA interference
  • invertebrates RNAi-based gene suppression was first demonstrated in nematodes (Fire et al, (1998) Nature, 391 :806-811; Timmons & Fire (1998) Nature, 395:854).
  • RNAi-based suppression of invertebrate genes using recombinant nucleic acid techniques has been reported in a number of species, including agriculturally or economically important pests from various insect and nematode taxa, such as: root-knot nematodes (Meloidogyne spp.), see Huang et al. (2006) Proc. Natl. Acad. Sci. USA, 103 : 14302-14306, doi: 10.1073/pnas.0604698103); cotton bollworm (Helicoverpa armigera), see Mao et al.
  • the present embodiments relate to control of insect species, especially flea beetle species that are economically or agriculturally important pests.
  • the compositions and methods disclosed herein comprise insecticidal polynucleotide molecules that are useful for controlling or preventing infestation by that insect species.
  • a polynucleotide-containing composition e. g. , a composition containing a dsRNA for suppressing a target gene
  • Other embodiments relate to methods for selecting insect target genes that are effective targets for RNAi-mediated control of an insect species.
  • a method for controlling an insect infestation of a plant comprising contacting the insect with a insecticidal polynucleotide comprising at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of the insect.
  • the insecticidal polynucleotide comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% complementarity with a fragment of a target gene of the insect.
  • the target gene is selected from the group consisting of Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpS21, RpS4, Rpn2, Rpn3, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase
  • the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • the insecticidal polynucleotide comprises a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide comprises at least one segment of 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975.
  • the insecticidal polynucleotide is a dsRNA comprising an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975.
  • the contacting of the insect is by oral delivery, or by non-oral contact, e. g. , by absorption through the cuticle, or through a combination of oral and non-oral delivery.
  • the insecticidal polynucleotide suppresses a target gene in the insect and stunts growth, development or reproduction by the insect, or kills the insect.
  • Several embodiments relate to a method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one insecticidal polynucleotide comprising at least one silencing element, wherein the at least one silencing element is essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, and wherein ingestion of the insecticidal polynucleotide by the insect results in mortality or stunting in the insect.
  • the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Protea
  • the target gene sequence is selected from the group consisting of SEQ ID NOs:l - 859.
  • the insecticidal polynucleotide comprises a sequence of about 95% to about 100% identity or complementarity to at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975.
  • the insecticidal polynucleotide is provided in the diet of the insect in the form of a plant or bacterial cell containing the polynucleotide, or as a synthetic polynucleotide molecule, or as a fermentation product (e. g. , a hairpin form of a dsRNA, produced in a bacterium).
  • the insecticidal polynucleotide is a single -stranded RNA molecule.
  • the insecticidal polynucleotide is a double- stranded RNA molecule.
  • the insecticidal polynucleotide is a single -stranded DNA molecule.
  • the insecticidal polynucleotide is a double-stranded DNA molecule.
  • the insecticidal polynucleotide is a RNA/DNA hybrid molecule.
  • an insecticidal composition comprising an insecticidally effective amount of a polynucleotide, wherein the polynucleotide comprises 18 or more contiguous nucleotides with about 95% to about 100% complementarity with a corresponding portion of a target gene of an insect that infests a plant.
  • the polynucleotide comprises 21 contiguous nucleotides with a sequence of 100% complementarity with a corresponding portion of the target gene.
  • the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI
  • the polynucleotide comprises 18 or more contiguous nucleotides with about 95% to about 100% complementarity with a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859 or a fragment thereof. In some embodiments, the polynucleotide comprises 21 contiguous nucleotides with a sequence of 100% complementarity with a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859 or a fragment thereof. In some embodiments, the polynucleotide is a single-stranded RNA molecule. In some embodiments, the polynucleotide is molecule is a dsRNA molecule.
  • the polynucleotide is a single- stranded DNA molecule. In some embodiments, the polynucleotide is a double-stranded DNA molecule. In some embodiments, the polynucleotide is a RNA/DNA hybrid molecule. In some embodiments, the polynucleotide comprises at least one segment (e. g. , an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, polynucleotide comprises at least one segment (e. g.
  • polynucleotide comprises at least one segment of 21 contiguous nucleotides (e. g. , an RNA strand or segment of an RNA strand) that is complementary or identical to a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal composition further comprises one or more of a carrier agent, a surfactant, an organosilicone, a cationic lipid, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator.
  • a carrier agent e.g. , a bacterially produced insecticidal protein.
  • Several embodiments relate to a method of providing a plant having improved resistance to an insect, comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA construct encodes an insecticidal polynucleotide comprising a sequence that is essentially identical or essentially complementary to a fragment of at least one target gene of the insect, and wherein ingestion of the insecticidal polynucleotide by the insect results in mortality or stunting in the insect.
  • the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Protea
  • the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • the insecticidal polynucleotide is single -stranded RNA (ssRNA).
  • the insecticidal polynucleotide is double- stranded RNA (dsR A), which may comprise single-stranded portions, such as a loop in a stem-loop structure.
  • the insecticidal polynucleotide is a single -stranded DNA molecule.
  • the insecticidal polynucleotide is a double -stranded DNA molecule.
  • the insecticidal polynucleotide is a RNA/DNA hybrid molecule. In some embodiments, the insecticidal polynucleotide comprises a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide comprises a sequence of at least 21 contiguous nucleotides that are complementarity or identical to a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975. In some
  • the insecticidal polynucleotide is an RNA (e. g. , an RNA strand or segment of an RNA strand) comprising at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof
  • a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an insecticidal polynucleotide comprising a sequence of about 95% to about 100% identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • a commercial unit of seed such as a bag
  • all or substantially all of the seeds comprise a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an insecticidal polynucleotide comprising a sequence of about 95% to about 100% identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide is single-stranded RNA (ssRNA).
  • the insecticidal polynucleotide is double-stranded RNA
  • the insecticidal polynucleotide is single -stranded DNA (ssDNA). In other embodiments, the insecticidal polynucleotide is double-stranded DNA (dsDNA), which may comprise single -stranded portions, such as a loop in a stem-loop structure. In some embodiments, the insecticidal polynucleotide is a hybrid RNA/DNA molecule.
  • the insecticidal polynucleotide comprisesan RNA strand or segment of an RNA strand comprising a sequence of about 95% to about 100% identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide comprises an RNA strand or segment of an RNA strandcomprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • compositions comprising a insecticidal polynucleotide as described herein, such as ssRNA, dsRNA, ssDNA, dsDNA or hybrid RNA/DNA formulations useful for topical application to a plant or substance in need of protection from an insect infestation; recombinant constructs and vectors useful for making transgenic plant cells and transgenic plants; formulations and coatings useful for treating plants (including plant seeds or propagatable parts such as tubers); plant seeds or propagatable parts such as tubers treated with or containing a polynucleotide as described herein as well as commodity products and foodstuffs produced from such plants; seeds, or propagatable parts (especially commodity products and foodstuffs having a detectable amount of a polynucleotide disclosed herein).
  • a insecticidal polynucleotide as described herein such as ssRNA, dsRNA, ssDNA, dsDNA or hybrid RNA/DNA formulations useful for topical application to a plant or
  • polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or the complement thereof.
  • Such antibodies are made by routine methods as known to one of ordinary skill in the art.
  • the present embodiments relate to methods and compositions for controlling insect pests, in particular the group of coleopteran insects commonly known as "flea beetles", of which there are several genera.
  • target genes identified as useful for designing insecticidal polynucleotide agents for preventing or treating flea beetle infestations, especially of commercially important plants.
  • the methods and compositions are especially useful for preventing or treating flea beetle infestations of commercially important Brassica species including species commercially used as oilseed, food, or livestock feed (e. g. , canola, rapeseed, turnips, and field mustard or turnip rape).
  • Such Brassica species include B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B.
  • septiceps B. nigra, B. narinosa, B. perviridus, B. facilitatorfortii, and B. fructiculosa.
  • sequences for suppressing one or more flea beetle target genes are also disclosed.
  • insecticidal polynucleotide agents that suppress flea beetle target genes.
  • insecticidal polynucleotides and recombinant DNA molecules and constructs useful in methods of controlling insect pests, especially flea beetles are provided.
  • Several embodiments relate to insecticidal compositions, as well as to transgenic plants resistant to infestation by insect pests.
  • RNA molecules for example, single -stranded RNA molecules, double-stranded RNA molecules, single-stranded DNA molecules, double-stranded DNA molecules, or hybrid RNA/DNA molecules for controlling insect pests and methods for identifying target genes that are likely to represent essential functions, making these genes preferred targets for RNAi -mediated silencing and control of insect pests.
  • Several embodiments relate to methods and compositions for inhibiting or controlling flea beetle infestation of a plant by inhibiting in the flea beetle the expression of one or more target gene selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, sh
  • RNA/DNA hybrid an insecticidal polynucleotide molecule, such as a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA hybrid, which comprises one or more segments comprising 18 or more contiguous nucleotides, for example 21 or more contiguous nucleotides, having 95% to about 100% (e.
  • the insecticidal polynucleotide comprises multiple segments each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%)
  • the insecticidal polynucleotide comprises at least 21 contiguous nucleotides having 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • the insecticidal polynucleotide comprises segments complementary to different regions of a target gene, or can comprise multiple copies of a segment.
  • the insecticidal polynucleotide comprises multiple segments, each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a different target gene; in this way multiple target genes, or multiple insect species, can be suppressed.
  • a insecticidal polynucleotide e.g., a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA hybrid
  • a insecticidal polynucleotide e.g., a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA hybrid
  • a insect target genes selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, R
  • an insecticidal polynucleotide having a length greater than that which is typical of naturally occurring regulatory small R As (such as endogenously produced siRNAs and mature miRNAs), e. g, thepolynucleotide is at least about 30 contiguous base-pairs in length. In some embodiments, the insecticidal polynucleotide has a length of between about 50 to about 500 base-pairs. In some embodiments, the insecticidal polynucleotide is at least 50 base pairs in length. In some embodiments, the insecticidal polynucleotide is formed from two separate, essentially complementary strands (e. g.
  • each strand is separately provided, or where each strand is encoded on a separate DNA molecule, or where the two strands are encoded on separate sections of a DNA and are separately transcribed or made separate, for example, by the action of a recombinase or nuclease), wherein at least one strand comprises a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide is double stranded and blunt-ended, e. g. , two separate, equal-length strands which form the double -stranded
  • the insecticidal polynucleotide is double stranded and has an overhang at one or both ends (termini), e. g. , two separate, unequal-length strands which form the double -stranded polynucleotide through intermolecular hybridisation; the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand.
  • the insecticidal polynucleotide is double stranded and has an overhang at one or both ends (termini), e. g. , two separate, unequal-length strands which form the double -stranded polynucleotide through intermolecular hybridisation; the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand.
  • polynucleotide comprises at least one stem -loop, e. g. , a single polynucleotide molecule that forms a double -stranded region through intramolecular hybridization adjacent to a "hairpin" secondary structure.
  • the insecticidal polynucleotide is formed from a single self- hybridizing hairpin, wherein one "arm" of the hairpin comprises a sequence of about 95% to about 100% (e. g.
  • self-hybridizing polynucleotides which form hairpins include polynucleotide molecules that comprise "spacer" nucleotides or a single -stranded "loop region" between the double-strand-forming complementary "arms" of sense sequence and anti-sense sequence.
  • such spacers or loops include nucleotides having a sequence unrelated (not complementary or identical to) the target gene corresponding to the double -stranded portion of the hairpin.
  • such spacers or loops include nucleotides having a sequence complementary or identical to the target gene. Examples of spacers or loops include those encoded by SEQ ID NOs:1719 - 1721.
  • the insecticidal polynucleotide comprises multiple stem-loops, with or without spacer nucleotides between each stem-loop.
  • the insecticidal polynucleotide comprises a modified stem-loop such as a "stabilized anti -sense” loop or a “stabilized sense” loop; see, e. g. , United States Patent Numbers 7,855,323 and 9,006,414, which are incorporated by reference in their entirety herein.
  • the insecticidal polynucleotide can be chemically synthesized (e. g., by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation.
  • the insecticidal polynucleotide can be chemically modified, e. g., to improve stability, ease of formulation, or efficacy.
  • the insecticidal polynucleotide molecule is provided in a microbial or plant cell that expresses the insecticidal polynucleotide (such as a hairpin form of a dsRNA), or in a microbial fermentation product.
  • RNA duplex that is formed by annealing of two separate RNA transcripts
  • United States Patent Numbers 7,855,323 and 9,006,414 to Huang et al which disclose recombinant DNA constructs including DNA encoding "stabilized anti- sense” transcripts which form a loop of anti -sense-oriented RNA for suppressing one or more target genes; recombinant DNA constructs can be designed to similarly encode "stabilized sense” transcripts which form a loop of sense-oriented RNA for suppressing one or more target genes.
  • Embodiments of the compositions comprising insecticical polynucleotides described herein further comprise one or more additional components or adjuvants, e. g. , a carrier agent, an encapsulation agent, an emulsifying agent, a surfactant, an organosilicone, a cationic lipid, a spreading agent, a photoprotective agent, a rainfastness agent, a polynucleotide herbicidal molecule, a non- polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a non-polynucleotide fungicide, a safener, a bait, an insect attractant, an insect pheromone, and an insect growth regulator.
  • these additional components or adjuvants are edible or digestible if ingested by a flea beetle.
  • the insecticidal polynucleotide disclosed herein are used in combination with a non-nucleotide pesticidal agent such as a small-molecule pesticidal agent or a proteinaceous pesticidal agent, either concurrently or sequentially.
  • a non-nucleotide pesticidal agent such as a small-molecule pesticidal agent or a proteinaceous pesticidal agent
  • non-nucleotide pesticidal agents include patatins, plant lectins, phytoecdysteroids, and bacterial insecticidal proteins (e. g.
  • insecticidal proteins from Bacillus thuringiensis, Xenorhabdus sp., Photorhabdus sp., Brevibacillus laterosporus (previously Bacillus later osporus), Lysinibacillus sphaericus (previously Bacillus sphaericus), Chromobacterium sp., Chromobacterium subtsugae, Paenibacillus sp., Paenibacillus lentimorbus , and Paenibacillus popilliae), a bacterium that produces an insecticidal protein, and an entomicidal bacterial species.
  • compositions comprising polynucleotides for flea beetle control such as the insecticidal polynucleotide described herein can further comprise, or can be used concurrently or sequentially with, conventional pesticides such as Spiromesifen, Spirodiclofen, Spirotetramat, Pyridaben, Tebufenpyrad, Tolfenpyrad, Fenpyroximate, Flufenerim, Pyrimidifen, Fenazaquin, Rotenone, Cyenopyrafen, Hydramethylnon, Acequinocyl, Fluacrypyrim, Aluminium phosphide, Calcium phosphide, Phosphine, Zinc phosphide, Cyanide, Diafenthiuron, Azocyclotin, Cyhexatin, Fenbutatin oxide, Propargite, Tetradifon, Bensultap, Thiocyclam, Thiosultap-sodium, Flonicamid,
  • Fenoxycarb Pyriproxyfen, Pymetrozine, Pyrifluquinazon, Chlorfenapyr, Tralopyril, B. t. ⁇ Bacillus thuringiensis) var. aizawai, B.t. var. israelensis, B.t. var. kurstaki, B.t. var. sphaericus, B.t. var.
  • Methiocarb Methomyl, Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiofanox, Triazamate,
  • compositions comprising insecticidal polynucleotide for flea beetle control in Brassica species, including canola further comprise, or are used concurrently or sequentially with, foliar sprays including one or more pesticides selected from the group consisting of Deltamethrin, Cypermethrin, Lambda- cyhalothrin, Permethrin, Carbaryl, Carbofuran, and Malathion, or seed treatments comprising one or more pesticides selected from the group consisting of Thiamethoxam, Imidacloprid, and Clothianadin.
  • foliar sprays including one or more pesticides selected from the group consisting of Deltamethrin, Cypermethrin, Lambda- cyhalothrin, Permethrin, Carbaryl, Carbofuran, and Malathion
  • seed treatments comprising one or more pesticides selected from the group consisting of Thiamethoxam, Imidacloprid
  • compositions comprising insecticidal polynucleotides for flea beetle control described herein can further comprise, or can be used concurrently or sequentially with, conventional fungicides such as bupirimate, dimethirimol, ethirimol, cyprodinil, pyrimethanil, mepanipyrim, fenpiclonil, fludioxonil; phenylamides, benalaxyl, furalaxyl, metalaxyl, i?-metalaxyl, ofurace, oxadixyl, benomyl, carbendazim, debacarb, fuberidazole, thiabendazole, chlozolinate, dichlozoline, iprodine, myclozoline, procymidone, vinclozolin, carboxin, fenfuram, flutolanil, mepronil, oxycarboxin, thifluzamide
  • conventional fungicides such
  • fluquinconazole flusilazole, flutriafol, hexaconazole, imazalil, imibenconazole, ipconazole, tebuconazole, tetraconazole, fenbuconazole, metconazole, myclobutanil, perfurazoate, penconazole, bromuconazole, pyrifenox, prochloraz, triadimefon, triadimenol, triflumizol, triticonazole, triforine, ancymidol, fenarimol or nuarimol, dodemorph, fenpropidin, fenpropimo ⁇ h, spiroxamine, tridemo ⁇ h, and fenhexamid.
  • compositions comprising insecticidal polynucleotides for flea beetle control in Brassica species, including canola further comprise, or are used concurrently or sequentially with, foliar sprays including one or more fungicides selected from the group consisting of Azoxystrobin, Bacillus subtilis strain QST 2808, Boscalid, Fluxopyroxad, Pyraclostrobin,
  • compositions comprising insecticidal polynucleotides for flea beetle described herein can further comprise, or can be used concurrently or sequentially with, conventional herbicides such as glyphosate, auxin-like herbicides such as dicamba, phosphinothricin, glufosinate, 2,2-dichloropropionic acid (Dalapon), acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates, and phthalide, bromoxynil,
  • conventional herbicides such as glyphosate, auxin-like herbicides such as dicamba, phosphinothricin, glufosinate, 2,2-dichloropropionic acid (Dalapon)
  • acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine,
  • cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop), sulfonamide herbicides, triazine herbicides, 5-methyltryptophan, aminoethyl cysteine, pyridazinone herbicides such as norflurazon, cyclopropylisoxazole herbicides such as isoxaflutole, protoporphyrinogen oxidase inhibitors, herbicidea containing an aryloxyalkanoate moiety, phenoxy auxins such as 2,4-D and dichlorprop, pyridyloxy auxins such as fluroxypyr and triclopyr, aryloxyphenoxypropionate s (AOPP) acetyl- coenzyme A carboxylase (ACCase) inhibitors such as haloxyfop, quizalofop, and diclofop, and 5- substituted phenoxyacetate protoporphyrin
  • compositions comprising insecticidal polynucleotides for flea beetle control in Brassica species, including canola further comprise, or are used concurrently or sequentially with, one or more post-emergence herbicides selected from the group consisting of Quizalofop, Sethoxydim, Clethodim, and Clopyralid.
  • the compositions comprising insecticidal polynucleotides for flea beetle control in herbicide-resistant Brassica species, including herbicide- resistant canola further comprise, or are used concurrently or sequentially with, one or more herbicides selected from the group consisting of Imazamox, Glyphosate, and Glufosinate.
  • compositions and methods disclosed are useful for inhibiting or controlling flea beetle infestation of a plant, such as a. Brassica species.
  • the compositions and methods are used to treat a growing plant, such as a field of Brassica plants.
  • Embodiments include compositions comprising insecticidal polynucleotides disclosed herein in a composition in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment composition.
  • compositions are applied to a surface of the plant in need of protection from or treatment for flea beetle infestations, or applied directly to the flea beetles, or provided in an ingestible form to the flea beetles.
  • a composition comprising insecticidal polynucleotides disclosed herein is applied directly to
  • ungerminated seeds such as ungerminated Brassica species seeds
  • seed treatment methods are disclosed in United States Patent Application 14/143,836, which is incorporated by reference in its entirety herein.
  • An embodiment includes a Brassica seed that is treated by directly contacting the seed with an insecticidal polynucleotide (such as a ssRNA, dsRNA, ssDNA, dsDNA or R A/DNA hybrid molecule) disclosed herein, followed by germination into a Brassica plant that exhibits increased resistance to flea beetle infestations.
  • insecticidal polynucleotide such as a ssRNA, dsRNA, ssDNA, dsDNA or R A/DNA hybrid molecule
  • nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction.
  • a given DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides.
  • T thymine
  • U uracil
  • a given first polynucleotide sequence whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), / ' . e. , a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base-pairs.
  • a first nucleic acid sequence is "operably” connected or “linked” with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter sequence is "operably linked” to DNA if the promoter provides for transcription or expression of the DNA.
  • operably linked DNA sequences are contiguous.
  • polynucleotide commonly refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to “oligonucleotides” (a polynucleotide molecule of 18- 25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules containing multiple nucleotides, including non-canonical nucleotides or chemically modified nucleotides as commonly practiced in the art; see, e. g, chemical modifications disclosed in the technical manual “RNA Interference (RNAi) and DsiRNAs", 2011 (Integrated DNA Technologies Coralville, IA).
  • RNAi RNA Interference
  • DsiRNAs Integrated DNA Technologies Coralville, IA
  • insecticidal polynucleotides as described herein comprise at least one segment of 18 or more contiguous nucleotides (or, in the case of double-stranded polynucleotides, at least 18 contiguous base-pairs) that are essentially identical or complementary to a fragment of equivalent size of the DNA of a target gene or the target gene's RNA transcript.
  • "at least 18 contiguous” means "from about 18 to about 10,000, including every whole number point in between” .
  • embodiments include compositions comprising oligonucleotides having a length of 18-25 nucleotides (18-mers, 19- mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or
  • nucleotides such as, but not limited to, polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the target gene).
  • polynucleotides can further be described in terms of one or more of the single -stranded components.
  • the insecticidal polynucleotides described herein can be single-stranded (ss) or double -stranded (ds).
  • “Double-stranded” refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double -stranded nucleic acid structure, generally under physiologically relevant conditions.
  • Embodiments include those wherein the polynucleotide is selected from the group consisting of sense single -stranded DNA (ssDNA), sense single -stranded RNA (ssRNA), double-stranded RNA (dsRNA), double -stranded DNA (dsDNA), a double -stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; a mixture of
  • the polynucleotide is double-stranded RNA of a length greater than that which is typical of naturally occurring regulatory small RNAs (such as endogenously produced siRNAs and mature miRNAs).
  • the insecticidal polynucleotide is double-stranded RNA of at least about 30 contiguous base-pairs in length.
  • the insecticidal polynucleotide is double- stranded RNA with a length of between about 50 to about 500 base-pairs.
  • the insecticidal polynucleotide can comprise components other than standard ribonucleotides, e. g., an embodiment is an RNA that comprises terminal deoxyribonucleotides.
  • Insecticidal polynucleotides of any size can be used, alone or in combination, in the various methods and compositions described herein.
  • a single insecticidal polynucleotide is used to make a composition (e. g., a composition for topical application).
  • a mixture or pool of different insecticidal polynucleotides is used; in such cases the insecticidal polynucleotides can be for a single target gene or for multiple target genes.
  • an insecticidal polynucleotide as described herein comprises naturally occurring nucleotides, such as those which occur in DNA and RNA.
  • the insecticidal polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides.
  • the insecticidal polynucleotide comprises non-canonical nucleotides such as inosine, thiouridine, or pseudouridine.
  • the insecticidal polynucleotide comprises chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication 2011/0152353, U.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with
  • modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e. g., fluorescein or rhodamine) or other label (e. g., biotin).
  • a fluorescent moiety e. g., fluorescein or rhodamine
  • other label e. g., biotin
  • recombinant as used to refer to a polynucleotide (such as insecticidal polynucleotide molecules or recombinant DNA constructs described herein), means that the polynucleotide is not a naturally occurring molecule, / ' . e. , that human intervention is required for the polynucleotide to exist.
  • a recombinant polynucleotide is produced using recombinant nucleic acid techniques, or by chemical synthesis, and can include combinations of sequences that do not occur in nature (e. g.
  • a recombinant polynucleotide can be biologically produced in a cell (such as a bacterial or plant or animal cell), for example, when that cell is transfected or transformed with a vector encoding the recombinant polynucleotide (e. g. , a vector encoding a hairpin form of a dsRNA, produced in a bacterium).
  • a recombinant polynucleotide can comprise sequences of nucleotides designed in silico using appropriate algorithms.
  • the insecticidal polynucleotides disclosed herein are generally designed to suppress or silence one or more genes ("target genes").
  • the term “gene” refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript, or that is a hereditable nucleic acid sequence.
  • a "gene” can include, but is not limited to, a promoter region, 5 ' untranslated regions, transcript encoding regions that can include intronic regions, 3 ' untranslated regions, or combinations of these regions.
  • the target genes can include coding or non-coding sequence or both.
  • the target gene has a sequence identical to or complementary to a messenger RNA, e. g. , in embodiments the target gene is a cDNA.
  • isolated refers to separating a molecule from other molecules normally associated with it in its native or natural state.
  • isolated thus may refer to a DNA molecule that has been separated from other DNA molecule(s) which normally are associated with it in its native or natural state.
  • Such a DNA molecule may be present in a recombined state, such as a recombinant DNA molecule.
  • DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques are considered isolated, even when integrated as a transgene into the chromosome of a cell or present with other DNA molecules.
  • insecticidally effective is meant effective in inducing a physiological or behavioural change in an insect (e. g. , adult or larval flea beetles) that infests a plant such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development.
  • insecticidally effective amount of the insecticidal polynucleotide, such as a dsRNA molecule to a plant improves the plant's resistance to infestation by the insect.
  • an insecticidally effective amount of the insecticidal polynucleotide to a crop plant improves yield (e. g. , increased biomass, increased seed or fruit production, or increased oil, starch, sugar, or protein content) of that crop plant, in comparison to a crop plant not treated with the insecticidal polynucleotide. While there is no upper limit on the concentrations and dosages of an insecticidal polynucleotide as described herein that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency and economy.
  • Non-limiting embodiments of effective amounts of an insecticidal polynucleotide include a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of an insecticidal polynucleotide in a liquid form sprayed on a plant, or from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied to a field of plants, or from about 0.001 to about 0.1 microgram per milliliter of polynucleotide in an artificial diet for feeding the insect.
  • compositions as described herein are topically applied to a plant
  • concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds.
  • a useful treatment for herbaceous plants using insecticidal polynucleotides as described herein is about 1 nanomole (nmol) of polynucleotides per plant, for example, from about 0.05 to 1 nmol polynucleotides per plant.
  • herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a single-stranded polynucleotide as described herein are applied. In certain embodiments, about 0.5 nmol to about 2 nmol of an insecticidal polynucleotide as described herein is applied.
  • a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of an insecticidal polynucleotide (or a single-stranded 21-mer) as described herein is applied.
  • a composition of about 0.5 to about 1.5 milligrams per milliliter of an insecticidal polynucleotide as described herein of about 50 to about 200 or more nucleotides is applied.
  • about 1 nmol to about 5 nmol of an insecticidal polynucleotide as described herein is applied to a plant.
  • a polynucleotide composition as topically applied to the plant comprises at least one polynucleotide as described herein at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter.
  • Very large plants, trees, or vines can require correspondingly larger amounts of insecticidal polynucleotides.
  • long insecticidal polynucleotide molecules that can be processed into multiple oligonucleotides e. g. , multiple oligonucleotides encoded by a single recombinant DNA molecule as disclosed herein
  • lower concentrations can be used.
  • Non-limiting examples of effective insecticidal polynucleotide treatment regimes include a treatment of between about 0.1 to about 1 nmol of polynucleotide molecule per plant, or between about 1 nmol to about 10 nmol of polynucleotide molecule per plant, or between about 10 nmol to about 100 nmol of polynucleotide molecule per plant.
  • Several embodiments relate to a method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one insecticidal polynucleotide comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:l - 859, and wherein ingestion of the insecticidal polynucleotide by the insect results in mortality or stunting in the insect.
  • These methods are useful for controlling insect infestations of a plant, for example for prevention or treatment of a flea beetle infestation of a crop plant, particularly commercially important Brassica species.
  • the at least one silencing element comprises a sequence having about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In embodiments, the at least one silencing element comprises 18 or more contiguous nucleotides with a sequence of 100%
  • the at least one silencing element comprises at least one segment of 18 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In embodiments, the at least one silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In embodiments the at least one silencing element comprises a sequence of about 95% to about 100% identity or
  • the insecticidal polynucleotide is provided in a microbial or plant cell that expresses the insecticidal polynucleotide, or in a microbial fermentation product, or is chemically synthesized.
  • the insecticidal polynucleotide comprises dsRNA.
  • the dsRNA is blunt-ended, or has an overhang at at least one terminus, or comprises at least one stem -loop.
  • the dsRNA is provided by convenient techniques commonly used.
  • the dsRNA is chemically synthesized, produced by expression in a
  • the dsRNA is made from naturally occurring ribonucleotides; in other embodiments the dsRNA is chemically modified.
  • the method is useful for causing mortality or stunting in insects that are pests of commercially important crop plants, such as an insect pest of a Brassica species.
  • the insect is a flea beetle.
  • the insect is a species of a genus selected from the group consisting of the genera. Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
  • the insect is a species selected from the group consisting oiAltica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (
  • the insect is a species selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes zonata (hop flea beetle).
  • Embodiments of the method include those in which the insecticidal polynucleotide is designed to silence a target gene in a genus- or species-specific manner, for example, wherein (a) the insect is a Phyllotreta species and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 551; (b) the insect is Phyllotreta atra (turnip flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296; (c) the insect is Phyllotreta cruciferae (canola flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532; (d) the insect is Phyllotreta striolata (striped flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:
  • Embodiments of the method also include those wherein (a) the insect is a. Phyllotreta species and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1410 or a fragment thereof; (b) the insect is Phyllotreta atra (turnip flea beetle) and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof; (c) the insect is Phyllotreta cruciferae (canola flea beetle) and insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731-1972, and 1974 or a fragment thereof; (d) the insect is Phyllotreta striolata (striped flea beetle) and the insecticidal polynucle
  • Embodiments of the method include those wherein at least one insecticidal polynucleotide is provided in a composition comprising the insecticidal polynucleotide , wherein the composition is applied to a surface of the insect or to a surface of a seed or plant in need of protection from infestation by the insect.
  • Embodiments of such compositions include those where the
  • composition is a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment.
  • the composition is formulated in a form that is ingested by the insect.
  • the composition further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organo silicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non- polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator.
  • the composition further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus ⁇ Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
  • Several embodiments relate to a method for controlling an insect infestation of a plant comprising contacting the plant and/or an insect that infests a plant with a insecticidal polynucleotide, wherein the insecticidal polynucleotide comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g.
  • a target gene of the insect selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta,
  • the insecticidal polynucleotide comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • controlling includes inducement of a physiological or behavioural change in an insect (adult or larvae or nymphs) such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development.
  • insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof, or the complement thereof.
  • the insect is a flea beetle, e. g., a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
  • a flea beetle e. g., a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
  • Hippuriphila Horaia, Hyphasis, Lipromima, Liprus, Longitarsus , Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona,
  • the insect is selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle).
  • the insect is selected from the group consisting of Phyllotreta armoraciae
  • Phyllotreta robusta garden flea beetle
  • Phyllotreta striolata striped flea beetle
  • Phyllotreta undulata Psylliodes chrysocephala
  • Psylliodes zonata hop flea beetle
  • the plant can be any plant that is subject to infestation by an insect that can be controlled by the insecticidal polynucleotides disclosed herein.
  • Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants".
  • the method is especially useful for controlling an insect infestation of an ornamental plant or a crop plant.
  • Various embodiments of the method include those wherein the plant is a plant in the family Brassicaceae, including a Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B.
  • the plant is selected from the group consisting of Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
  • Glycine max Linum usitatissimum
  • Zea mays Carthamus tinctorius
  • Helianthus annuus Helianthus annuus
  • Nicotiana tabacum Arabidopsis thaliana
  • Betholettia excelsa, Ricinus communis
  • Methods include those developed for specific flea beetle pests for a given plant, e. g. , wherein the plant is a potato plant and the insect is Epitrix cucumeris (potato flea beetle).
  • specific target genes are identified as targets for RNAi-mediated control in a given insect species.
  • Various embodiments of the method include those wherein (a) the insect is Phyllotreta atra (turnip flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296; (b) the insect is Phyllotreta cruciferae (canola flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532; (c) the insect is Phyllotreta striolata (striped flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551; or (d) the insect is Psylliodes chrysocephala and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859.
  • specific insecticidal polynucleotides are developed for specific target genes in a given insect species.
  • Embodiments of the method include those wherein (a) the insect is Phyllotreta atra (turnip flea beetle) and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof; (b) the insect is Phyllotreta cruciferae (canola flea beetle) and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731-1972, and 1974 or a fragment thereof; (c) the insect is Phyllotreta striolata (striped flea beetle) and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:1392
  • chrysocephala and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:1411 - 1718 or a fragment thereof.
  • the method includes contacting an insect, such as a flea beetle, with an insecticidal polynucleotide .
  • an insect such as a flea beetle
  • Embodiments include contacting via oral delivery to the insect, or non-oral delivery to the insect, or a combination of oral and non-oral delivery to the insect.
  • Embodiments include contacting insects in the adult stage, or in larval stages, or in the egg stage. In some embodiments, contacting results in mortality (death) or stunting (growth stunting or decrease in or cessation of metamorphosis stage development) of the insect, thereby preventing or treating infestation of the plant by the insect.
  • contacting results in inducement of a physiological or behavioural change in an insect (adult or larvae or nymphs) that results in a decreased ability of the insect to infest or damage a plant, for example, a decrease in reproductive capacity, or a decrease in or cessation of feeding behavior or movement.
  • the contacting comprises application of a composition comprising one or more insecticidal polynucleotide to a surface of the insect or to a surface of the plant infested by the insect.
  • the composition can comprise or be in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment.
  • the contacting comprises providing the insecticidal polynucleotide in a composition that further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator.
  • the contacting comprises providing the one or more insecticidal polynucleotides in a composition that further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus ⁇ Bacillus sphaericus), Brevibacillus laterosporus ⁇ Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus
  • the contacting comprises providing one or more insecticidal polynucleotides in a composition that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait.
  • compositions can further comprise one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non- polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator.
  • compositions can further comprise at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species,
  • a pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal
  • the combination of the insecticidal polynucleotide and the non-polynucleotide pesticidal agent provides a level of insect control that is greater than the sum of the effects of the insecticidal polynucleotide and the non-polynucleotide pesticidal agent components if tested separately.
  • insecticidally effective amount of an insecticidal polynucleotide such as a ssR A, dsR A, ssDNA, dsDNA or hybrid RNA/DNA molecule, wherein the insecticidal polynucleotide comprises at least 18 or more contiguous nucleotides with a sequence of about 95% to about 100% ⁇ e. g. , about 95%, about
  • the insecticidal polynucleotide comprises at least 18 or more contiguous nucleotides having about 95% to about 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments the insecticidal polynucleotide comprises at least 21 contiguous nucleotides having 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In embodiments, the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insect is a flea beetle, e. g., a species of a genus selected from the group consisting of the genemAltica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis,
  • Luperomorpha Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and Zipangia.
  • the insect is selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle).
  • Altica ambiens alder flea beetle
  • Altica canadensis prairie flea beetle
  • the insect is selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes zonata (hop flea beetle).
  • the insecticidal composition is useful for treating a plant or area in the vicinity of a plant to provide protection or treatment from insects, especially flea beetles.
  • a related aspect is a plant treated with an insecticidal composition as described herein, or a seed of the treated plant, wherein the plant exhibits improved resistance to the insect (e. g. , improved resistance to flea beetles).
  • the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not treated with the insecticidal composition.
  • yield (oilseed biomass or oil content) in canola or oilseed rape plants is improved by application of an insecticidally effective amount of a insecticidal polynucleotidetargetting one or more genes identified from Phyllotreta cruciferae (canola flea beetle); in particular embodiments, the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532.
  • the plant can be any plant that is subject to infestation by an insect that can be controlled by the insecticidal composition. Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants".
  • the method is especially useful for controlling an insect infestation of an ornamental plant or a crop plant.
  • Various embodiments include those wherein the plant is a plant in the family Brassicaceae, including a Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournamentfortii, and B. fructiculosa.
  • the plant is selected from the group consisting of Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
  • Glycine max Linum usitatissimum
  • Zea mays Carthamus tinctorius
  • Helianthus annuus Helianthus annuus
  • Nicotiana tabacum Arabidopsis thaliana
  • Betholettia excelsa, Ricinus communis
  • the insecticidal composition is developed for specific flea beetle pests for a given plant, e. g. , where the plant is a potato plant and the insect is Epitrix cucumeris (potato flea beetle). In some embodiments, the insecticidal composition is developed for specific target genes in a given insect species.
  • insecticidal composition include those wherein (a) the insect is Phyllotreta atra (turnip flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296; (b) the insect is Phyllotreta cruciferae (canola flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532; (c) the insect is Phyllotreta striolata (striped flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551; or (d) the insect is Psylliodes chrysocephala and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859
  • the insecticidal polynucleotide molecule of use in this method is provided as an isolated insecticidal polynucleotide molecule (not part of an expression construct, e. g. , lacking additional elements such as a promoter or terminator sequences).
  • Such insecticidal polynucleotide molecules can be relatively short, such as single- or double-stranded RNA, DNA or hybrid RNA/DNA molecules of between about 18 to about 300 or between about 50 to about 500 nucleotides (for single -stranded polynucleotides) or between about 18 to about 300 or between about 50 to about 500 base-pairs (for double-stranded polynucleotides).
  • the polynucleotide is a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment.
  • the insecticidal composition is provided in a form that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait.
  • the insecticidal compositions can further comprise one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator.
  • the insecticidal compositions can further comprise at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a
  • phytoecdysteroid a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus ⁇ Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus , and Paenibacillus popilliae.
  • the combination of the insecticidal polynucleotide molecule and the non-polynucleotide pesticidal agent provides a level of insect control that is greater than the sum of the effects of the insecticidal polynucleotide molecule and the non- polynucleotide pesticidal agent components if tested separately.
  • Embodiments of the compositions optionally comprise the appropriate stickers and wetters required for efficient foliar coverage as well as UV protectants to protect insecticidal polynucleotides, such as dsR As, from UV damage. Such additives are commonly used in the bioinsecticide industry and are known to those skilled in the art.
  • Compositions for soil application can comprise granular formulations that serve as bait for insect larvae.
  • Embodiments include a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator.
  • compositions may comprise a "transfer agent", an agent that, when combined with a composition comprising an insecticidal polynucleotide as disclosed herein that is topically applied to the surface of an organism, enables the polynucleotide to enter the cells of that organism.
  • a transfer agent can be incorporated as part of the composition comprising a insecticidal polynucleotide as disclosed herein, or can be applied prior to, contemporaneously with, or following application of the composition comprising an insecticidal polynucleotide as described herein.
  • a transfer agent is an agent that improves the uptake of an insecticidal polynucleotide by an insect.
  • a transfer agent is an agent that conditions the surface of plant tissue, e. g , seeds, leaves, stems, roots, flowers, or fruits, to permeation by an insecticidal polynucleotide into plant cells.
  • the transfer agent enables a pathway for a polynucleotide through cuticle wax barriers, stomata, and/or cell wall or membrane barriers into plant cells.
  • Suitable transfer agents include agents that increase permeability of the exterior of the organism or that increase permeability of cells of the organism to polynucleotides.
  • Suitable transfer agents include a chemical agent, or a physical agent, or combinations thereof.
  • Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof.
  • application of an insecticidal polynucleotide and a transfer agent optionally includes one or more of an incubation step, a neutralization step (e. g.
  • Suitable transfer agents can be in the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition, or can cause the polynucleotide composition to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition.
  • transfer agents include counter-ions or other molecules that are known to associate with nucleic acid molecules, e.
  • cationic lipids inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations.
  • transfer agents include organic solvents such as DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents miscible with water or that dissolve
  • Embodiments of transfer agents include naturally derived or synthetic oils with or without surfactants or emulsifiers, e. g. , plant-sourced oils, crop oils (such as those listed in the 9 th Compendium of Herbicide Adjuvants, publicly available on-line at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N- pyrrolidine.
  • naturally derived or synthetic oils with or without surfactants or emulsifiers e. g. , plant-sourced oils, crop oils (such as those listed in the 9 th Compendium of Herbicide Adjuvants, publicly available on-line at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N- pyrrolidine.
  • Embodiments of transfer agents include organosilicone preparations.
  • a suitable transfer agent is an organosilicone preparation that is commercially available as SILWET L- 77® brand surfactant having CAS Number 27306-78-1 and EPA Number: CAL. REG NO. 5905-50073- AA, and currently available from Momentive Performance Materials, Albany, New York.
  • SILWET L-77® brand surfactant organosilicone preparation is used as transfer agent in the form of a spray treatment (applied prior to, contemporaneously with, or following application of the composition comprising an insecticidal polynucleotide as disclosed herein) of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e.
  • 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of a polynucleotide as disclosed herein into plant cells from a topical application on the surface.
  • One embodiment includes a composition that comprises a polynucleotide and a transfer agent including an organosilicone preparation such as Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent).
  • organosilicone preparation such as Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01, 0.015, 0.0
  • One embodiment includes a composition that comprises a polynucleotide and a transfer agent including SILWET L-77® brand surfactant in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about ⁇ % t by weight (wt percent).
  • a transfer agent including SILWET L-77® brand surfactant in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about ⁇ % t by weight (wt percent).
  • Organosilicone compounds useful as transfer agents for use in compositions and methods disclosed herein include, but are not limited to, compounds that include: (a) a trisiloxane head group that is covalently linked to, (b) an alkyl linker including, but not limited to, an ⁇ -propyl linker, that is covalently linked to, (c) a polyglycol chain, that is covalently linked to, (d) a terminal group.
  • Trisiloxane head groups of such organosilicone compounds include, but are not limited to,
  • Alkyl linkers can include, but are not limited to, an ⁇ -propyl linker.
  • Polyglycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Polyglycol chains can include a mixture that provides an average chain length "n" of about "7.5". In certain
  • the average chain length "n" can vary from about 5 to about 14.
  • Terminal groups can include, but are not limited to, alkyl groups such as a methyl group.
  • Organosilicone compounds useful as transfer agents for use in compositions and methods disclosed herein include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane.
  • An example of a transfer agent for use in compositions and methods disclosed herein is Compound I:
  • Organo silicone compounds useful as transfer agents for use in compositions and methods disclosed herein are used, e. g. , as freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent).
  • wt percent percent by weight
  • Embodiments of transfer agents include one or more salts such as ammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate, provided in or used with a composition comprising an insecticidal polynucleotide disclosed herein.
  • ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w/v), or about 1% to about 3% (w/v), or about 2% (w/v).
  • the composition comprising an insecticidal polynucleotide comprises an ammonium salt at a concentration greater or equal to 300 millimolar.
  • the composition comprising an insecticidal polynucleotide comprises an organosilicone transfer agent in a concentration of about 0.015 to about 2 percent by weight (wt percent) as well as ammonium sulfate at concentrations from about 80 to about 1200 millimolar or about 150 millimolar to about 600 millimolar.
  • Embodiments of transfer agents include a phosphate salt.
  • Phosphate salts useful in a composition comprising an insecticidal polynucleotide include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts.
  • the composition comprising an insecticidal polynucleotide includes a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar.
  • the composition comprising an insecticidal polynucleotide includes a phosphate salt in a range of about 1 millimolar to about 25 millimolar or in a range of about 5 millimolar to about 25 millimolar.
  • the composition comprising an insecticidal polynucleotide includes sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a sodium phosphate salt in a range of about 1 millimolar to about 25 millimolar or in a range of about 5 millimolar to about 25 millimolar.
  • the composition comprising an insecticidal polynucleotide includes a sodium phosphate salt in a range of about 10 millimolar to about 160 millimolar or in a range of about 20 millimolar to about 40 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a sodium phosphate buffer at a pH of about 6.8.
  • Embodiments of transfer agents include surfactants and/or effective molecules contained therein.
  • Surfactants and/or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organo silicone surfactants.
  • the composition comprising an insecticidal polynucleotide is formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules. Non-limiting examples include, tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine.
  • the composition comprising an insecticidal polynucleotide is formulated with a non-polynucleotide herbicide e. g. , glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben, and TBA, glufosinate, auxin-like herbicides including phenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylic acid herbicide, and benazolin-ethyl herbicide, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides.
  • a non-polynucleotide herbicide e. g. , glyphosate, auxin
  • polynucleotide is formulated with a non-polynucleotide pesticide, e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.
  • a non-polynucleotide pesticide e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.
  • Several embodiments relate to a method of providing a plant having improved resistance to an insect, comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA construct comprises DNA encoding an insecticidal polynucleotidecomprising a sequence of at least 21 contiguous nucleotides that are essentially identical or essentially
  • the DNA construct comprises DNA encoding an insecticidal polynucleotide comprising a sequence essentially identical or essentially complementary to a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • the DNA construct comprises DNA encoding an insecticidal polynucleotide having about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID
  • the recombinant DNA construct further comprises a heterologous promoter operably linked to the DNA encoding an insecticidal polynucleotide, wherein the heterologous promoter is functional in a plant cell.
  • heterologous refers to nucleic acid sequences that are not usually operably linked in a native or naturally occurring genome; by “heterologous promoter” is meant that the promoter is not natively operably linked with the DNA encoding an insecticidal polynucleotide. Promoters functional in a plant cell include those listed under the heading "Promoters”.
  • the recombinant DNA construct is expressed in the plant by means of transgenic expression or transient expression.
  • the method further comprises expression in the plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus ⁇ Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae,
  • the pesticidal agent can be expressed from the same recombinant DNA construct that comprises the DNA encoding an insecticidal polynucleotide, or from a different recombinant DNA construct.
  • a related aspect is a plant having improved resistance to an insect (e. g. , improved resistance to flea beetles), or the seed of such a plant, wherein the plant is provided by the method comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA construct comprises DNA encoding an insecticidal polynucleotide having a sequence essentially identical or essentially complementary to a fragment of at least one target gene of the insect, wherein the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, Rp
  • the recombinant DNA construct comprises DNA encoding an insecticidal polynucleotide comprising a sequence essentially identical or essentially complementary to a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
  • the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not expressing the recombinant DNA construct.
  • yield (oilseed biomass or oil content) in canola or oilseed rape plants is improved by expressing in the canola or oilseed rape plants an insecticidal polynucleotide, such as a dsRNA molecule, targetting one or more genes of Phyllotreta cruciferae (canola flea beetle), e. g. , wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532.
  • the plant can be any plant that is subject to infestation by an insect that can be controlled by expressing in the plant the recombinant DNA construct according to this method.
  • Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants".
  • the method is especially useful for providing an ornamental plant or a crop plant with improved resistance to flea beetles.
  • Various embodiments of the method include those wherein the plant is a plant in the family Brassicaceae, including a Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B.
  • the plant is selected from the group consisting of Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgar e, and Triticum aestivum.
  • the method provides a potato plant
  • Embodiments of the method provide a plant having improved resistance to one or more flea beetle species, e. g., a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis,
  • a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela
  • Lipromima Liprus, Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia,
  • the insect is selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle).
  • Altica ambiens alder flea beetle
  • Altica canadensis prairie flea beetle
  • the method provides a plant having improved resistance to an insect selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes zonata (hop flea beetle).
  • the method is developed for specific target genes in a given insect species.
  • a plant having improved resistance to Phyllotreta atra (turnip flea beetle), wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296, is provided.
  • a plant having improved resistance to Phyllotreta cruciferae (canola flea beetle), wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532, is provided.
  • a plant having improved resistance to Phyllotreta striolata (striped flea beetle), wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551, is provided.
  • a plant having improved resistance to Psylliodes chrysocephala, wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859 is provided.
  • a plant having improved resistance to Phyllotreta atra that expresses from a recombinant DNA construct a dsRNA strand comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof is provided.
  • a plant having improved resistance to Phyllotreta cruciferae that expresses from a recombinant DNA construct an insecticidal polynucleotide comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:1156 - 1391 , 1731-1972, and 1974 or a fragment thereof is provided.
  • a plant having improved resistance to Phyllotreta striolata that expresses from a recombinant DNA construct an insecticidal polynucleotide comprising a strand comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975 or a fragment thereof is provided.
  • a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an insecticidal polynucleotide transcript comprising a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity to at least 21 contiguos nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide transcript comprises a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide transcript forms dsRNA double-stranded insecticidal polynucleotide.
  • the insecticidal polynucleotide transcript is a dsRNA comprising an RNA strand comprising at least one segment of 18 or more contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide transcript is a dsRNA comprising an RNA strand comprising at least one segment of 21 contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the insecticidal polynucleotide transcript is a dsRNA comprising at least one RNA strand comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a dsRNA comprising an RNA strand comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript forms single-stranded insecticidal polynucleotide.
  • the insecticidal polynucleotide transcript is a ssRNA comprising at least one segment of 18 or more contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising at least one segment of 21 contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising at least one segment of 21 contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID
  • the insecticidal polynucleotide transcript is a ssRNA comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • the heterologous promoter is functional for expression of the insecticidal polynucleotide transcript in a bacterium.
  • the bacterium is selected from the group consisting of Escherichia coli, Bacillus species, Pseudomonas species,
  • the recombinant DNA construct comprises a heterologous promoter that is functional in a plant cell.
  • the recombinant DNA construct is contained in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector.
  • the recombinant DNA construct is integrated into a plant chromosome or plastid, e. g. , by stable transformation.
  • transgenic plant cell comprising in its genome the recombinant DNA construct, and a transgenic plant comprising such a transgenic plant cell.
  • Transgenic plant cells and plants are made by methods known in the art, such as those described under the heading "Making and Using Transgenic Plant Cells and Transgenic Plants". Further aspects include a commodity product produced from such a transgenic plant, and transgenic progeny seed or
  • Suitable plants in which the methods and compositions disclosed herein can be used include, but are not limited to, cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas including canola and oilseed rape, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, fava bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut),
  • Additional plant species of interest are Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum. Additional Construct Elements
  • Embodiments of the polynucleotides and nucleic acid molecules disclosed herein can comprise additional elements, such as promoters, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e. g. , to express a transgene such as an insecticidal protein or selectable marker) or non -coding sequences (e. g. , to express additional suppression elements).
  • additional elements such as promoters, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e. g. , to express a transgene such as an insecticidal protein or selectable marker) or non -coding sequences (e. g. , to express additional suppression elements).
  • an aspect provides a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
  • a recombinant DNA construct comprising a promoter operably linked to DNA encoding: (a) an RNA transcript comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof, and (b) an aptamer, is stably integrated into the plant's genome from where RNA transcripts comprising the RNA aptamer and the RNA silencing element are expressed in cells of the plant; the aptamer serves to guide the RNA silencing element to a desired location in the cell.
  • inclusion of one or more recognition sites for binding and cleavage by a small RNA e. g. , by a miRNA or an siRNA that is expressed only in a particular cell or tissue
  • a small RNA e. g. , by a miRNA or an siRNA that is expressed only in a particular cell or tissue
  • Promoters of use in the compositions and methods disclosed herein are functional in the cell in which the construct is intended to be transcribed. Generally these promoters are
  • heterologous promoters as used in recombinant constructs, i. e. , they are not in nature found to be operably linked to the other nucleic elements used in the constructs.
  • the promoter is selected from the group consisting of a constitutive promoter, a spatially specific promoter, a temporally specific promoter, a developmentally specific promoter, and an inducible promoter.
  • the promoter is a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
  • Non-constitutive promoters suitable for use with the recombinant DNA constructs disclosed herein include spatially specific promoters, temporally specific promoters, and inducible promoters.
  • Spatially specific promoters can include organelle-, cell-, tissue-, or organ-specific promoters (e. g. , a plastid-specific, a root-specific, a pollen-specific, or a seed-specific promoter for expression in plastids, roots, pollen, or seeds, respectively).
  • organelle-, cell-, tissue-, or organ-specific promoters e. g. , a plastid-specific, a root-specific, a pollen-specific, or a seed-specific promoter for expression in plastids, roots, pollen, or seeds, respectively.
  • a seed-specific, embryo- specific, aleurone-specific, or endosperm-specific promoter is especially useful.
  • Temporally specific promoters can include promoters that tend to promote expression during certain developmental stages in a plant's growth cycle, or during different times of day or night, or at different seasons in a year.
  • Inducible promoters include promoters induced by chemicals or by environmental conditions such as, but not limited to, biotic or abiotic stress (e. g. , water deficit or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection).
  • MicroR A promoters are useful, especially those having a temporally specific, spatially specific, or inducible expression pattern;
  • An expression-specific promoter can also include promoters that are generally constitutively expressed but at differing degrees or “strengths" of expression, including promoters commonly regarded as “strong promoters” or as “weak promoters”.
  • Promoters include the following examples: an opaline synthase promoter isolated from T-DNA of Agrobacterium; a cauliflower mosaic virus 35S promoter; enhanced promoter elements or chimeric promoter elements such as an enhanced cauliflower mosaic virus (CaMV) 35 S promoter linked to an enhancer element (an intron from heat shock protein 70 of Zea mays); root specific promoters such as those disclosed in U.S. Patents 5,837,848; 6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S. Patent 6,433,252; a promoter for a plant nuclear gene encoding a plastid-localized aldolase disclosed in U. S.
  • Patent Application Publication 2004/0216189 cold- inducible promoters disclosed in U.S. Patent 6,084,089; salt-inducible promoters disclosed in U. S. Patent Number 6, 140,078; light-inducible promoters disclosed in U.S. Patent 6,294,714; pathogen- inducible promoters disclosed in U.S. Patent 6,252, 138; and water deficit-inducible promoters disclosed in U.S. Patent Application Publication 2004/0123347 Al . All of the above-described patents and patent publications disclosing promoters and their use, especially in recombinant DNA constructs functional in plants are incorporated herein by reference.
  • Plant vascular- or phloem-specific promoters of interest include a rolC or rolA promoter of Agrobacterium rhizogenes, a promoter of a.
  • Agrobacterium tumefaciens T-DNA gene 5 the rice sucrose synthase RSsl gene promoter, a Commelina yellow mottle badnavirus promoter, a coconut foliar decay virus promoter, a rice tungro bacilliform virus promoter, the promoter of a pea glutamine synthase GS3A gene, a invCD l 1 1 and invCD 141 promoters of a potato invertase genes, a promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by Kertbundit et al.
  • VAHOX1 promoter region a pea cell wall invertase gene promoter, an acid invertase gene promoter from carrot, a promoter of a sulfate transporter gene Sultrl;3, a promoter of a plant sucrose synthase gene, and a promoter of a plant sucrose transporter gene.
  • Promoters suitable for use with a recombinant DNA construct or polynucleotide disclosed herein include polymerase II ("pol II") promoters and polymerase III ("pol III") promoters.
  • RNA polymerase II transcribes structural or catalytic RNAs that are usually shorter than 400 nucleotides in length, and recognizes a simple run of T residues as a termination signal; it has been used to transcribe siRNA duplexes (see, e. g. , Lu et al. (2004) Nucleic Acids Res., 32:e l71).
  • Pol II promoters are therefore preferred in certain embodiments where a short RNA transcript is to be produced from a recombinant DNA construct.
  • the recombinant DNA construct includes a pol II promoter to express an R A transcript flanked by self-cleaving ribozyme sequences (e. g , self-cleaving hammerhead ribozymes), resulting in a processed RNA, such as a single-stranded RNA that binds to the transcript of the flea beetle target gene, with defined 5 ' and 3 ' ends, free of potentially interfering flanking sequences.
  • An alternative approach uses pol III promoters to generate transcripts with relatively defined 5' and 3' ends, / ' . e. , to transcribe an RNA with minimal 5' and 3' flanking sequences.
  • Pol III promoters e.
  • U6 or HI promoters are preferred for adding a short AT-rich transcription termination site that results in 2 base-pair overhangs (UU) in the transcribed RNA; this is useful, e. g , for expression of siRNA-type constructs.
  • UU base-pair overhangs
  • pol III promoters for driving expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol, 20: 446-448.
  • Baculovirus promoters such as baculovirus polyhedrin and p 10 promoters are known in the art and commercially available; see, e.
  • the promoter element can include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologues thereof but that can regulate expression of a gene.
  • “gene independent" regulatory sequences include naturally occurring or artificially designed RNA sequences that comprise a ligand-binding region or aptamer (see “Aptamers", below) and a regulatory region (which can be cz ' s -acting). See, for example, Isaacs et al. (2004) Nat. Biotechnol, 22:841-847, Bayer and Smolke (2005) Nature Biotechnol, 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol, 5:451-463, Davidson and Ellington (2005) Trends
  • riboregulators could be selected or designed for specific spatial or temporal specificity, for example, to regulate translation of DNA that encodes a silencing element for suppressing a target gene only in the presence (or absence) of a given concentration of the appropriate ligand.
  • a riboregulator that is responsive to an endogenous ligand (e. g , jasmonic acid or salicylic acid) produced by the plant when under stress (e.
  • abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as attach by pests or pathogens
  • the level of endogenous ligand increases to a level sufficient for the riboregulator to begin transcription of the DNA that encodes a silencing element for suppressing a target gene.
  • the recombinant DNA construct or polynucleotide disclosed herein comprisies a transgene transcription unit.
  • a transgene transcription unit comprises DNA sequence encoding a gene of interest, e. g. , a natural protein or a heterologous protein.
  • a gene of interest can be any coding or non-coding sequence from any species (including, but not limited to, non- eukaryotes such as bacteria, and viruses; fungi, protists, plants, invertebrates, and vertebrates.
  • genes of interest are genes encoding one or more proteins conferring resistance to an herbicide and genes encoding at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a.
  • Bacillus thuringiensis insecticidal protein Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and an insecticidal protein produced by any of Lysinibacillus sphaericus ⁇ Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
  • the transgene transcription unit can further comprise 5' or 3' sequence or both as required for transcription of the transgene.
  • the recombinant DNA construct or polynucleotide comprises DNA encoding a spliceable intron.
  • intron is generally meant a segment of DNA (or the RNA transcribed from such a segment) that is located between exons (protein-encoding segments of the DNA or corresponding transcribed RNA), wherein, during maturation of the messenger RNA, the intron present is enzymatically "spliced out” or removed from the RNA strand by a cleavage/ligation process that occurs in the nucleus in eukaryotes.
  • intron is also applied to non-coding DNA sequences that are transcribed to RNA segments that can be spliced out of a maturing RNA transcript, but are not introns found between protein-coding exons.
  • spliceable sequences that that have the ability to enhance expression in plants (in some cases, especially in monocots) of a downstream coding sequence; these spliceable sequences are naturally located in the 5' untranslated region of some plant genes, as well as in some viral genes (e. g. , the tobacco mosaic virus 5' leader sequence or "omega” leader described as enhancing expression in plant genes by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638).
  • expression-enhancing introns can be artificially inserted in the 5' untranslated region of a plant gene between the promoter but before any protein-coding exons.
  • expression-enhancing introns include, but are not limited to, a maize alcohol dehydrogenase (Z -Adhl), a maize Bronze-1 expression-enhancing intron, a rice actin 1 (Os-Actl) intron, a Shrunken- 1 (Sh-1) intron, a maize sucrose synthase intron, a heat shock protein 18 (hsplS) intron, and an 82 kilodalton heat shock protein (hsp82) intron.
  • Z -Adhl maize alcohol dehydrogenase
  • Os-Actl rice actin 1
  • Shrunken- 1 Sh-1
  • hsplS heat shock protein 18
  • hsp82 82 kilodalton heat shock protein
  • Patents 5,593,874 and 5,859,347 describe methods of improving recombinant DNA constructs for use in plants by inclusion of an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the non-translated leader positioned 3' from the gene promoter and 5' from the first protein-coding exon.
  • hsp70 heat shock protein
  • the recombinant DNA construct or polynucleotide comprises DNA encoding additional gene suppression element for suppressing a target gene other than a flea beetle target gene.
  • the target gene to be suppressed can include coding or non-coding sequence or both.
  • Suitable gene suppression elements are described in detail in U. S. Patent Application Publication 2006/0200878, which disclosure is specifically incorporated herein by reference, and include one or more of:
  • DNA that transcribes to RNA for suppressing the gene to be suppressed by forming a single double -stranded RNA and comprises multiple serial anti-sense DNA segments that are anti- sense to at least one segment of the gene to be suppressed and multiple serial sense DNA segments that are at least one segment of the gene to be suppressed;
  • multiple double strands of RNA and comprises multiple anti -sense DNA segments that are anti-sense to at least one segment of the gene to be suppressed and multiple sense DNA segments that are at least one segment of the gene to be suppressed, and wherein the multiple anti-sense DNA segments and the multiple sense DNA segments are arranged in a series of inverted repeats;
  • an intron is used to deliver a gene suppression element in the absence of any protein-coding exons (coding sequence).
  • an intron such as an expression-enhancing intron (preferred in certain embodiments) is interrupted by embedding within the intron a gene suppression element, wherein, upon transcription, the gene suppression element is excised from the intron.
  • protein-coding exons are not required to provide the gene suppressing function of the recombinant DNA constructs disclosed herein.
  • the recombinant DNA construct or polynucleotide comprises DNA encoding a transcription regulatory element.
  • Transcription regulatory elements include elements that regulate the expression level of the recombinant DNA construct (relative to its expression in the absence of such regulatory elements). Examples of suitable transcription regulatory elements include riboswitches (cis- or fra «s-acting), transcript stabilizing sequences, and miRNA recognition sites, as described in detail in U. S. Patent Application Publication 2006/0200878, specifically incorporated herein by reference .
  • the recombinant DNA constructs disclosed herein can be stacked with other recombinant DNA for imparting additional traits (e. g. , in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like) for example, by expressing or suppressing other genes.
  • additional traits e. g. , in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like
  • Constructs for coordinated decrease and increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 Al, specifically incorporated by reference.
  • the transgenic plant contains recombinant DNA further comprising a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct for flea beetle control is preferably effected with concurrent transcription of the gene expression element.
  • the transgenic plant expresses DNA encoding a insecticidal polynucleotide transcript as disclosed herein for suppression of a flea beetle target gene, and also expresses DNA encoding a non-nucleotide pesticidal agent such as a small-molecule pesticidal agent or a proteinaceous pesticidal agent; such DNAs can be stacked in a single recombinant construct or expression cassette, or alternatively can be expressed from discrete recombinant constructs or expression cassettes.
  • non-nucleotide pesticidal agents include patatins, plant lectins, phytoecdysteroids, and bacterial insecticidal proteins (e. g.
  • the transgenic plant expresses DNA encoding a recombinant RNA transcript as disclosed herein for suppression of a flea beetle target gene, and also expresses DNA encoding one or more proteins conferring tolerance to an herbicide.
  • proteins conferring tolerance to an herbicide include 5 -enolpyruvylshikimate-3 -phosphate synthases (EPSPS; see, e. g. , U. S. Patent Numbers 5,627,061, 5,633,435 RE39247, 6,040,497, and 5,094,945, and PCT International Application Publications WO04074443 and WO04009761), glyphosate oxidoreductase (GOX; U. S.
  • EPSPS 5 -enolpyruvylshikimate-3 -phosphate synthases
  • Patent Number 5,463, 175) glyphosate decarboxylase
  • PCT International Application Publication WO05003362 U. S. Patent Number 7,405,347, and U. S. Patent Application Publication 2004/0177399
  • glyphosate-N-acetyl transferase GAT; U. S. Patent Number 7,714, 188 conferring tolerance to glyphosate
  • dicamba monooxygenase conferring tolerance to auxin -like herbicides such as dicamba (U. S. Patent Number 7, 105,724)
  • phosphinothricin acetyltransferase (pat or bar) conferring tolerance to phosphinothricin or glufosinate (U. S. Patent Number 5,646,024)
  • 2,2- dichloropropionic acid dehalogenase conferring tolerance to 2,2-dichloropropionic acid (Dalapon) (PCT International Application Publication
  • acetohydroxyacid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide
  • acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide
  • haloarylnitrilase (Bxn) for conferring tolerance to bromoxynil
  • modified acetyl-coenzyme A carboxylase for conferring tolerance to cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop)
  • acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimid
  • Patent Number 6,414,222 dihydropteroate synthase (sul I) for conferring tolerance to sulfonamide herbicides (U. S. Patent Number 5,719,046); 32 kDa photosystem II polypeptide (psbA) for conferring tolerance to triazine herbicides (Hirschberg et al , 1983, Science, 222: 1346-1349); anthranilate synthase for conferring tolerance to 5-methyltryptophan (U. S.
  • Patent Number 4,581,847 dihydrodipicolinic acid synthase (dap A) for conferring to tolerance to aminoethyl cysteine (PCT International Application Publication W0891 1789); phytoene desaturase (crtl) for conferring tolerance to pyridazinone herbicides such as norflurazon (Japan Patent JP06343473); hydroxyphenylpyruvate dioxygenase, a 4- hydroxyphenylacetic acid oxidase and a 4-hydroxyphenylacetic 1 -hydrolase (US Patent 7,304,209) for conferring tolerance to cyclopropylisoxazole herbicides such as isoxaflutole (U. S. Patent Number 6,268,549); modified protoporphyrinogen oxidase I (protox) for conferring tolerance to
  • protoporphyrinogen oxidase inhibitors U. S. Patent Number 5939602
  • aryloxyalkanoate dioxygenase AAD-1 for conferring tolerance to an herbicide containing an aryloxyalkanoate moiety
  • herbicides examples include phenoxy auxins (such as 2,4- D and dichlorprop), pyridyloxy auxins (such as fluroxypyr and triclopyr), aryloxyphenoxypropionates (AOPP) acetylcoenzyme A carboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, and diclofop), and 5- substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors (such as pyraflufen and flumiclorac).
  • phenoxy auxins such as 2,4- D and dichlorprop
  • pyridyloxy auxins such as fluroxypyr and triclopyr
  • AOPP aryloxyphenoxypropionates
  • ACCase acetylcoenzyme A carboxylase
  • ACCase acetylcoenzyme A carboxylase
  • haloxyfop such as haloxyfo
  • the recombinant DNA constructs disclosed herein can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage.
  • Transgenic plants can be derived from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood- or pulp-producing trees, vegetable plants, fruit plants, and ornamental plants.
  • plants of interest include grain crop plants (such as wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees; vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, the brassicas (for example, cabbages, kales, mustards, and other leafy brass
  • Specific plant species of interest in which a recombinant DNA construct is transcribed to provide resistance to flea beetles are plants in the family Brassicaceae, including the Brassica species B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. facilitatorfortii, and B. fructiculosa.
  • Additional plant species of interest in which a recombinant DNA construct is transcribed to provide resistance to flea beetles are Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
  • a transgenic plant having in its genome a recombinant DNA construct as disclosed herein exhibits increased resistance to an insect infestation, specifically increased resistance to a flea beetle infestation.
  • the transgenic plant expresses a recombinant DNA construct for flea beetle control that is stacked with other recombinant DNA for imparting additional traits
  • the transgenic plant has at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of:
  • the transgenic plant is characterized by: improved tolerance of abiotic stress (e. g. , tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or of biotic stress (e. g. , crowding, allelopathy, or wounding); by a modified primary metabolite (e. g. , fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition; a modified secondary metabolite (e. g. , alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition; a modified trace element (e. g.
  • abiotic stress e. g. , tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels
  • biotic stress e. g. , crowding, allelopathy, or wounding
  • carotenoid e. g. , beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls
  • vitamin e. g. , tocopherols
  • improved yield e. g. , improved yield under non-stress conditions or improved yield under biotic or abiotic stress
  • improved ability to use nitrogen, phosphate, or other nutrients modified agronomic characteristics (e. g. , delayed ripening; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to "green snap" of stems; modified photoperiod response); modified growth or reproductive characteristics (e.
  • transgenic seed, or seed produced by the transgenic plant has modified primary metabolite (e. g. , fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, a modified secondary metabolite composition, a modified trace element, carotenoid, or vitamin composition, an improved harvest, storage, or processing quality, or a combination of these.
  • modified primary metabolite e. g. , fatty acid, oil, amino acid, protein, sugar, or carbohydrate
  • a modified secondary metabolite composition e. e. fatty acid, oil, amino acid, protein, sugar, or carbohydrate
  • a modified trace element e.g., fatty acid, oil, amino acid, protein, sugar, or carbohydrate
  • carotenoid e.g., a modified trace element, carotenoid, or vitamin composition
  • it can be desirable to change levels of native components of the transgenic plant or seed of a transgenic plant for example, to decrease levels of an allergenic protein or glycoprotein or of
  • This example illustrates non-limiting embodiments of coding DNA sequences useful as target genes for controlling insect species and for making compositions for controlling insects and insect-resistant transgenic plants, and identifies insecticidal polynucleotide sequences useful for controlling insect species. More specifically, embodiments of target genes identified by name (annotation) and sequence identifier (SEQ ID NO.) for controlling flea beetles are provided in SEQ ID NOs:l - 859, and embodiments of dsRNA sequences ranging in size from 135 to 352 base pairs and designed to suppress these target genes are provided in SEQ ID NOs.:860 - 1718.
  • RNA sequences are provided for the anti-sense strand of the dsRNA in 5' to 3' direction.
  • RNA -mediated suppression of the corresponding target gene identified in Table 1 are useful for controlling insects, especially flea beetles, including the source species from which the target genes in Table 1 were identified.
  • RNA-mediated suppression of one or more of the target genes provided in Table 1, or use of one or more of the dsRNAs provided in Table 1, is useful for causing mortality or stunting, or otherwise controlling, target insect species in the following genera: Altica,
  • Anthobiodes Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus,
  • compositions comprising a dsRNA for suppression of one or more of the target genes provided in Table 1 (e. g.
  • a composition comprising an effective amount of one or more of the dsRNAs provided in Table 1) are useful for controlling at least one of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle), thus preventing or treating plant infestation by these species.
  • RNA-mediated suppression of one or more of the target genes provided in Table 1, or use of one or more of the dsRNAs provided in Table 1, is useful for causing mortality or stunting in flea beetle species in the genera Phyllotreta and Psylliodes, thus preventing or treating plant infestation by these species.
  • RNA-mediated suppression of one or more of the target genes provided in Table 1, or use of one or more of the dsRNAs provided in Table 1, is useful for causing mortality or stunting in at least one flea beetle species selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes Veronica
  • RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs: 1 - 296 is used to cause mortality or stunting in Phyllotreta atra (turnip flea beetle) adults or larvae, for example, by contacting Phyllotreta atra adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1155.
  • RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs:297 - 532 is used to cause mortality or stunting in Phyllotreta cruciferae (canola flea beetle) adults or larvae, for example, by contacting Phyllotreta cruciferae adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731-1972, and 1974.
  • RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs:533 - 551 is used to cause mortality or stunting in Phyllotreta striolata (striped flea beetle) adults or larvae, for example, by contacting Phyllotreta striolata adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975.
  • RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs:552 - 859 is used to cause mortality or stunting in Psylliodes chrysocephala adults or larvae, for example, by contacting Psylliodes chrysocephala adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:1411 - 1718.
  • Plants which can be protected by such infestation by transgenic expression or topical application of one or more of the dsRNAs provided in Table 1 include any plant species or variety that is subject to infestation by flea beetles, especially plants of economic importance, including ornamental plants and crop plants.
  • Embodiments of such plants include plants in the family Brassicaceae (mustard family), such as a plant in the genus Brassica including, for example, one of the following: B. napus (rapeseed, including cultivars such as canola and rutabaga), B. juncea (Indian mustard), B. carinata (Abyssinian mustard), B. rapa (turnip), B.
  • oleracea wild cabbage, including domesticated cultivars such as, kale, cabbage, broccoli, cauliflower, brussels sprouts, etc.
  • B. rupestris brown mustard
  • B. septiceps eventop mustard
  • B. nigra black mustard
  • B. narinosa broadbeaked mustard
  • the target plants may include, but not limited to, one of the following: Glycine max (soybean), Linum usitatissimum (linseed/flax), Zea mays (maize), Carthamus tinctorius (safflower), Helianthus annuus (sunflower), Nicotiana tabacum (tobacco), Arabidopsis thaliana, Betholettia excelsa (Brazil nut), Ricinus communis (castor bean), Cocos nucifera (coconut), Coriandrum sativum (coriander), Gossypium spp.
  • compositions comprising an effective amount of one or more of the dsR As provided in Table 1 for topical treatment of a plant to be treated for, or be protected from, flea beetle infestation.
  • Another aspect includes a recombinant DNA construct encoding at least one strand of at least one the dsRNAs provided in Table 1 for transgenic expression in a plant that has improved resistance to flea beetle infestation, in comparison to a plant not expressing such a construct.
  • This example illustrates non-limiting embodiments of testing the efficacy of dsRNA sequences and validating the dsR A's utility for suppressing expression of target genes for controlling insect species. More specifically this example illustrates a method comprising contacting an insect, such as a flea beetle adult or larva, with one or more dsRNAs designed to cause stunting or mortality in the insect. Other embodiments include methods where the dsRNA is delivered to the insect by oral delivery (e. g. , on or in a food material ingested by the insect), or through non-oral delivery (e. g. , delivery through the insect's cuticle, or delivery by contacting an egg of the insect).
  • oral delivery e. g. , on or in a food material ingested by the insect
  • non-oral delivery e. g. , delivery through the insect's cuticle, or delivery by contacting an egg of the insect.
  • a feeding assay is used to determine efficacy of a dsRNA in causing stunting or mortality in insects, such as flea beetles.
  • a single discriminating dose for example, 100 nanograms/milliliter
  • a negative control dsRNA such as a dsRNA targetting green fluorescent protein (GFP) is also included in the assay.
  • GFP green fluorescent protein
  • Each dsRNA is coated evenly onto 1.0 centimeter diameter canola leaf discs and placed in multiwell trays, with 2 male and 2 female adult flea beetles or 4 flea beetle larvae per well. Every 24 hours for a set period (e. g. , 2 weeks), new, freshly-coated leaves are provided. Stunting and mortality are scored periodically (e. g., daily, or every 2 or 3 days).
  • dsRNAs that show efficacy in this single-dose assay are tested further. Using a similar protocol, varying doses of dsRNAs are tested, as described above, to determine the LC50 dose for each of the active dsR As.
  • Bioassays include 12 - 24 insects per dose, performed in triplicate. Stunting and mortality is assessed over a 2 week period, scored on every third day.
  • the dsRNA sequences that are confirmed to be effective in suppressing a target gene in a sequence -specific manner are useful for identifying efficacious R A delivery agents and formulations.
  • the insecticidal activity of formulations containing dsRNA can be optimized by various techniques, such as modifying the chemical entities in the formulation or modifying the ratio of the chemical components in the formulation.
  • Non-limiting examples of delivery agents and formulations are provided in Example 5.
  • This example illustrates non-limiting embodiments of methods for validating dsRNA efficacy for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect. More specifically this example illustrates methods for testing dsRNA for efficacy in preventing or treating flea beetle infestations in whole plants.
  • Polynucleotides such as the dsRNA sequences described in Examples 1 and 2) that have been confirmed to be effective in suppressing a target gene in a sequence-specific manner are further evaluated in whole plant assays.
  • the polynucleotides e. g., anti-sense RNA, dsRNA
  • the polynucleotides are applied directly to the insect surface (e. g. by spraying or dusting).
  • the polynucleotides are provided to the insect in an insect diet (e. g. , in a bacterial or plant cell expressing a polynucleotide such as a hairpin form of a dsRNA, or in an artificial bait containing a polynucleotide). Stunting and mortality are scored periodically, as described in Example 2.
  • the polynucleotide is applied in a foliar application through aerial or terrestrial spraying or dusting or chemigation on the leaf surface to control early season damage from the adult stage of the life cycle, or applied as a seed treatment to control larval or adult stages of the insect life cycle, or applied as a soil in-furrow or drench application to control larval or adult stages of the insect life cycle.
  • An example of a foliar testing regime includes treating the plant immediately after emergence from the ground and evaluating foliar damage caused by adult flea beetles 1 - 2 weeks after plant emergence. For in-furrow or seed treatment similar timing for damage evaluation is followed.
  • the polynucleotides are generally designed to modulate expression by inducing regulation or suppression of an insect target gene and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence an insect target gene or cDNA (e. g., SEQ ID NOs:l - 859) or to the sequence of RNA transcribed from an insect target gene, which can be coding sequence or non-coding sequence.
  • This example describes non-limiting techniques useful in the design and selection of polynucleotides to modulate expression of an insect target gene. Selection of Effective Polynucleotides by "Tiling"
  • Polynucleotides of use in the compositions and methods disclosed herein need not be of the full length of a target gene, and in many embodiments are of much shorter length in comparison to the target gene.
  • An example of a technique that is useful for selecting effective polynucleotides for insect control is "tiling", or evaluation of polynucleotides corresponding to adjacent or partially overlapping segments of a target gene.
  • Effective polynucleotides can be identified by "tiling" gene targets in selected length fragments, e. g. , fragments of 200 - 300 nucleotides in length, with partially overlapping regions, e. g, of about 25 nucleotides, along the length of the target gene.
  • polynucleotides are designed to correspond to (have a nucleotide identity or complementarity with) regions that are unique to the target gene; the selected region of the target gene can include coding sequence or non- coding sequence (e. g. , promoter regions, 3 ' untranslated regions, introns and the like) or a combination of both.
  • the multiple target gene sequences are aligned and polynucleotides designed to correspond to regions with high sequence homology in common among the multiple targets.
  • the multiple target gene sequences are aligned and polynucleotides designed to correspond to regions with no or low sequence homology in common among the multiple targets.
  • polynucleotides e.g, anti -sense single -stranded RNAs, dsRNAs, anti-sense single-stranded DNAs, dsDNAs
  • polynucleotides capable of inducing suppression are designed for each of the target genes listed in Table 1 as follows.
  • polynucleotides each of 200 - 300 nucleotides in length and comprising a sequence complementary to a fragment of a target gene having a sequence selected from SEQ ID NOs:l - 859 are designed so that each polynucleotide's sequence overlaps about 25 nucleotides of the next adjacent polynucleotide's sequence, in such a way that the multiple polynucleotides in combination cover the full length of the target gene.
  • double- stranded polynucleotides can be designed by providing pairs of sense and anti -sense polynucleotides, each pair of polynucleotides overlapping the next adjacent pair of polynucleotides.
  • the polynucleotides are tested by any convenient means for efficacy in silencing the insect target gene. Examples of a suitable test include the bioassays described herein in the working Examples. Another test involves the topical application of the polynucleotides either directly to individual insects or to the surface of a plant to be protected from an insect infestation.
  • One desired result of treatment with a polynucleotide as disclosed herein is prevention or control of an insect infestation, e. g. , by inducing in an insect a physiological or behavioural change such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development.
  • Another desired result of treatment with a polynucleotide as disclosed herein is provision of a plant that exhibits improved resistance to an insect infestation.
  • the tiling procedure can be repeated, if desired.
  • a polynucleotide found to provide desired activity can itself be subjected to a tiling procedure.
  • multiple overlapping polynucleotides are designed, each of 50 - 60 nucleotides in length and comprising a sequence complementary to the fragment of a target gene having a sequence selected from SEQ ID NOs:l - 859 for which a single polynucleotide of 300 nucleotides was found to be effective. Additional rounds of tiling analysis can be carried out, where polynucleotides as short as 18, 19, 20, or 21 nucleotides are tested.
  • Effective polynucleotides of any size can be used, alone or in combination, in the various methods disclosed herein.
  • a single polynucleotide is used to make a composition (e. g. , a composition for topical application, or a recombinant DNA construct useful for making a transgenic plant).
  • a mixture or pool of different polynucleotides is used; in such cases the polynucleotides can be for a single target gene or for multiple target genes.
  • a polynucleotide is designed to target different regions of the target gene, e. g. , an insecticidal polynucleotide can comprise multiple segments that correspond to different exon regions of the target gene, and "spacer" nucleotides which do not correspond to a target gene can optionally be used in between or adjacent to the segments.
  • Polynucleotides can be designed or their sequence optimised using thermodynamic considerations. For example, insecticidal polynucleotides can be selected based on the thermodynamics controlling hybridization between one nucleic acid strand (e. g. , a polynucleotide or an individual siRNA) and another (e. g. , a target gene transcript)
  • one nucleic acid strand e. g. , a polynucleotide or an individual siRNA
  • another e. g. , a target gene transcript
  • polynucleotide or at least one strand of a double-stranded polynucleotide has sufficient identity or complementarity to the target gene or to the RNA transcribed from a target gene (e. g. , the transcript) to suppress expression of a target gene (e. g. , to effect a reduction in levels or activity of the target gene transcript and/or encoded protein).
  • a target gene e. g. , the transcript
  • Polynucleotides need not have 100 percent identity or
  • the polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In some embodiments, the polynucleotide or a portion thereof is designed to be exactly identical to, or exactly complementary to, a sequence of 21 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially identical"
  • polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides of the target gene or an RNA transcribed from the target gene.
  • an "essentially complementary" polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides of the target gene or an RNA transcribed from the target gene.
  • an "essentially complementary" polynucleotide has 100 percent sequence
  • Polynucleotides comprising mismatches to the target gene or transcript can be used in certain embodiments of the compositions and methods disclosed herein.
  • the polynucleotide comprises at least 18 or at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript.
  • a polynucleotide of 19 contiguous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript.
  • a polynucleotide of 20 or more nucleotides that comprises a contiguous 19 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript.
  • a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript.
  • a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript.
  • mismatches In designing polynucleotides with mismatches to a target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et al. (2005) Nucleic Acids Res. , 33 : 1671-1677.
  • mismatches in 19 base-pair overlap regions are located at the low tolerance positions 5, 7, 8 or 1 1 (from the 5 ' end of a 19-nucleotide target), at medium tolerance positions 3, 4, and 12-17(from the 5 ' end of a 19-nucleotide target), and/or at the high tolerance positions at either end of the region of complementarity, e. g , positions 1, 2, 18, and 19 (from the 5' end of a 19-nucleotide target) as described by Du et al. (2005) Nucleic Acids Res. , 33 : 1671-1677.
  • the polynucleotides comprise additional nucleotides (e.g., for reasons of stability or for convenience in cloning or synthesis).
  • the polynucleotide is a single -stranded RNA comprising an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 and further comprising an additional 5' G or an additional 3' C or both, adjacent to the segment.
  • the polynucleotide is a dsR A comprising an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 and further comprising an additional 5' G or an additional 3' C or both, adjacent to the segment.
  • the polynucleotide is a double -stranded RNA comprising additional nucleotides to form an overhang, for example, a dsRNA comprising 2 deoxyribonucleotides to form a 3' overhang.
  • a a polynucleotide with a sequence complementary to the target gene and which is responsible for an observed suppression of the target gene is embedded in "neutral" sequence, e. g. , inserted into additional nucleotides that have no sequence identity or complementarity to the target gene.
  • Neutral sequence can be desirable, e. g. , to increase the overall length of a polynucleotide.
  • a 21-base-pair dsRNA corresponding to a target gene in Table 1 and found to provide control of an insect infestation is embedded in neutral sequence of an additional 39 base pairs, thus forming a polynucleotide of about 60 base pairs.
  • a single 21-base-pair polynucleotide is found to be efficacious when embedded in larger sections of neutral sequence, e. g. , where the total polynucleotide length is from about 60 to about 300 base pairs.
  • At least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 is embedded in larger sections of neutral sequence to provide an efficacious insecticidal polynucleotide.
  • segments from multiple sequences selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 are embedded in larger sections of neutral sequence to provide an efficacious insecticidal polynucleotide.
  • polynucleotides disclosed herein e. g., single-strand RNA, dsRNA, single-strand DNA, or dsDNAcomprising a sequence selected from SEQ ID NOs:860 - 1718 and 1722 - 1975, or active fragments thereof
  • non-polynucleotide pesticidal agents e.g., single-strand RNA, dsRNA, single-strand DNA, or dsDNAcomprising a sequence selected from SEQ ID NOs:860 - 1718 and 1722 - 1975, or active fragments thereof
  • Routine insect bioassays such as the bioassays described herein in the working Examples are useful for defining dose-responses for larval mortality or growth inhibition using combinations of the polynucleotides disclosed herein and one or more non-polynucleotide pesticidal agents (e. g. , a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus
  • a patatin e.g. , a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus
  • This example illustrates non-limiting embodiments of methods of testing the efficacy of insecticidal polynucleotides in flea beetles. More specifically this example illustrates a method including oral delivery of polynucleotides to flea beetles, resulting in stunting or mortality in the flea beetles.
  • P. cruciferae were collected from a canola field where no pesticides had been applied in the previous 3 months.
  • Three dsRNAs (SEQ ID NOs:1169, 1193, and 1392) targeting Phyllotreta genes and one negative control dsRNA targeting GFP were tested on groups of 30 P.
  • dsRNAs were resuspended in water and applied to 6 millimeter leaf discs (55 ⁇ 6 milligrams each) at a discriminating dose of 50 nanograms dsR A/milligram leaf tissue, which were fed to groups of 5 flea beetles.
  • Leaf discs with freshly applied dsRNA were replaced every other day, and the number of surviving individuals was recorded over a 2-week period.
  • a low non-specific mortality rate was observed in the negative-control insect groups (3 out of 30 insects dying over 2 weeks, or 10% non-specific mortality). Mortality was observed beginning at day 4 and continuing through the 2 week period. Specific mortality was observed for all dsRNA treatments (Table 2).
  • the overall mortality rate for the negative controls was -4% (likely due to handling injuries) over the 12-day observation period.
  • Eight of the dsRNAs (indicated by a mortality rating of +++) caused 90 - 100% mortality at the highest dose and were still highly effective (80% or higher mortality) at the lowest dose tested.
  • Some dsRNAs (indicated by a mortality rating of ++) induced mortality in a proportion of the insects at the highest dose, but were less effective at lower doses ( ⁇ 20% mortality).
  • *+++ rating indicates high (>80%) mortalities for all three doses; ++ rating indicates high mortalities for the highest dose, and within 40 to 70% mortality with the lower two doses.
  • Other techniques for delivering these or similar insecticidal polynucleotides include applying the polynucleotides directly to the insect surface (e. g. by spraying or dusting), or providing the polynucleotide sto the insect in a diet or bait (e. g., in a bacterial or plant cell expressing a dsRNA, or in an artificial bait containing the dsRNA).
  • a hairpin version of the Phyllotreta insecticidal polynucleotide with the sequence SEQ ID NO: 1169 is designed; this hairpin version is encoded by the DNA sequence SEQ ID NO:1722, which contains, in 5' to 3' order, anti-sense sequence (nucleotide positions 1 - 267), loop sequence (nucleotide positions 268 - 373) which does not contain matches to Phyllotreta sequences, and sense sequence (nucleotide positions 374 - 640).
  • This DNA sequence is expressed as a single-stranded RNA transcript, where the anti-sense and sense regions anneal to form the double-stranded "stem" region of the hairpin.
  • the construct is expressed in a bacterium, such as E. coli; the resulting dsRNA hairpin produced in the bacterium is provided to flea beetles as a crude or purified fermentation product, or in the form of the bacterial cells.
  • Similar constructs are designed encoding dsRNAs having modified stem-loops, such as "stabilized anti-sense” or “stabilized sense” versions, which contain stabilized loops formed by an extended anti-sense or sense sequence, respectively, of sequence corresponding to the intended target gene.
  • modified stem-loops such as “stabilized anti-sense” or “stabilized sense” versions, which contain stabilized loops formed by an extended anti-sense or sense sequence, respectively, of sequence corresponding to the intended target gene.
  • This example discloses embodiments related to polynucleotide molecules having a nucleotide sequence containing specific modifications such as nucleotide substitutions.
  • Embodiments of such modifications include modified polynucleotides that provide improved sequence discrimination between the intended target gene of the insect pest of interest, and genetic sequences of other, non- target species.
  • Selected dsRNAs identified in Table 1 were screened for unintended sequence matches to a sequence of at least 19 contiguous nucleotides identified in a non -target gene or a non-target organism (NTO, e. g., Apis mellifera, Bombus impatiens and B. terrestris; Coleomegilla spp.; Danaus plexippus; Homo sapiens; Megachile rotundata; Mus musculus; and Brassica rapa).
  • NTO non-target gene or a non- target organism
  • Nucleotide changes are made in an original polynucleotide sequence to eliminate contiguous sequence matches to a non-target gene or non-target organism.
  • modified polynucleotide sequences are provided by SEQ ID NO:1723, which corresponds to SEQ ID NO:1393 (which targets the same flea beetle gene as does the insecticidal polynucleotide of SEQ ID NO: 1392), and SEQ ID NO:1724, which corresponds to SEQ ID NO:1169.
  • Embodiments discloses embodiments related to polynucleotide molecules having a nucleotide sequence for silencing a target gene in more than one species.
  • Embodiments include dsRNA sequences of at least 21 contiguous nucleotides identified as having 100% complementarity or identity to more than one ortholog of a target gene.
  • Table 4 provides a list of sequences, each at least 21 contiguous nucleotides in length and identified by the sequence's coordinates in a dsRNA for one flea beetle species, wherein the identical sequence is also found in a dsRNA for a different flea beetle species. These sequences are useful to design insecticidal polynucleotides against multiple species in which the target sequence co- occurs.
  • SEQ ID NO:1186 (targetting a Phyllotreta cruciferae COPI alpha target gene, SEQ ID NO:327) contains five sequences of at least 21 contiguous nucleotides at positions 1 - 71, 88 - 116, 136 - 209, 238 - 266, and 274 - 296, all of which match a sequence in SEQ ID NOs:882 and 888 (targetting a. Phyllotreta atra COPI alpha target genes, SEQ ID NOs:23 and 29, respectively); these five sequences are therefore useful in targeting a gene in the two Phyllotreta species.

Abstract

Disclosed herein are methods for controlling invertebrate pest infestations, particularly in plants; compositions and insecticidal polynucleotides useful in such methods; and plants having improved resistance to the invertebrate pests. More specifically, insecticidal polynucleotides and methods of use thereof for modifying the expression of genes in an insect pest, particularly through RNA interference are disclosed.

Description

COMPOSITIONS AND METHODS FOR CONTROLLING INSECT PESTS
CROSS-REFERENCE TO RELATED APPLICATION AND INCORPORATION OF SEQUENCE LISTING
[0001] This application claims priority to U.S. Provisional Patent Application No.
62/287,080, entitled "Compositions and Methods for Controlling Insect Pests," filed January 26, 2016, which is incorporated in its entirety. The sequence listing contained in the file
"P34170WO01_SL.TXT" (2,289,028 bytes, created on January 26, 2017) is filed herewith and is incorporated by reference in its entirety herein. FIELD
[0002] Disclosed herein are methods for controlling invertebrate pest infestations, particularly in plants, compositions and insecticidal polynucleotides useful in such methods, and plants having improved resistance to the invertebrate pests. More specifically, insecticidal polynucleotides and methods of use thereof for modifying the expression of genes in an insect pest, particularly through RNA interference are disclosed. Pest species of interest include flea beetles, such as Phyllotreta spp. and Psylliodes spp.
BACKGROUND
[0003] Commercial crops are often the targets of attack by invertebrate pests such as insects. RNA interference (RNAi, RNA -mediated gene suppression) is an approach that shows promise for use in environmentally friendly pest control. In invertebrates, RNAi-based gene suppression was first demonstrated in nematodes (Fire et al, (1998) Nature, 391 :806-811; Timmons & Fire (1998) Nature, 395:854). Subsequently, RNAi-based suppression of invertebrate genes using recombinant nucleic acid techniques has been reported in a number of species, including agriculturally or economically important pests from various insect and nematode taxa, such as: root-knot nematodes (Meloidogyne spp.), see Huang et al. (2006) Proc. Natl. Acad. Sci. USA, 103 : 14302-14306, doi: 10.1073/pnas.0604698103); cotton bollworm (Helicoverpa armigera), see Mao et al. (2007) Nature Biotechnol, 25 : 1307-1313, doi: 10.1038/nbtl352; Western corn rootworm (Diabrotica virgifera LeConte), see Baum et al. (2007) Nature Biotechnol, 25: 1322-1326, doi: 10.1038/nbtl359; sugar beet cyst nematode (Heterodera schachtii), see Sindhu et al. (2008) J Exp. Botany, 60:315-324, doi: 10.1093/jxb/ern289; mosquito (Aedes aegypti), see Pridgeon et al. (2008) J. Med. Entomol , 45:414-420, doi: full/ 10.1603/0022- 2585%282008%2945%5B414%3ATAADRK%5D2.0.CO%3B2; fruit flies {Drosophila melanogaster), flour beetles (Tribolium castaneum), pea aphids (Acyrthosiphon pisum), and tobacco hornworms (Manduca sexta), see Whyard et al. (2009) Insect Biochem. Mol. Biol, 39:824-832,
doi: 10.1016/j .ibmb.2009.09.00; diamondback moth (Plutella xylostella), see Gong et al. (2011) Pest Manag. Sci. , 67: 514-520, doi: 10.1002/ps.2086; green peach aphid (Myzus persicae), see Pitino et al. (2011) PLoS ONE, 6:e25709, doi: 10.1371/journal.pone.0025709; brown planthopper (Nilaparvata lugens), see Li et al. (2011) Pest Manag. Sci., 67: 852-859, doi: 10.1002/ps.2124; and whitefly (Bemisia tabaci), see Upadhyay et al. (2011) J Biosci. , 36: 153-161, doi: 10.1007/sl2038-011-9009-1.
SUMMARY
[0004] The present embodiments relate to control of insect species, especially flea beetle species that are economically or agriculturally important pests. The compositions and methods disclosed herein comprise insecticidal polynucleotide molecules that are useful for controlling or preventing infestation by that insect species. Several embodiments described herein relate to a polynucleotide-containing composition (e. g. , a composition containing a dsRNA for suppressing a target gene) that is topically applied to an insect species or to a plant, plant part, or seed to be protected from infestation by an insect species. Other embodiments relate to methods for selecting insect target genes that are effective targets for RNAi-mediated control of an insect species.
[0005] Several embodiments relate to a method for controlling an insect infestation of a plant comprising contacting the insect with a insecticidal polynucleotide comprising at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a target gene of the insect. In some embodiments, the insecticidal polynucleotide comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% complementarity with a fragment of a target gene of the insect. In some embodiments, the target gene is selected from the group consisting of Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpS21, RpS4, Rpn2, Rpn3, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S 14, and 60S ribosomal protein L13. In some embodiments, the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide comprises a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide comprises at least one segment of 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975. In some embodiments, the insecticidal polynucleotide is a dsRNA comprising an RNA strand with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975. In various embodiments, the contacting of the insect is by oral delivery, or by non-oral contact, e. g. , by absorption through the cuticle, or through a combination of oral and non-oral delivery. In some embodiments, the insecticidal polynucleotide suppresses a target gene in the insect and stunts growth, development or reproduction by the insect, or kills the insect. [0006] Several embodiments relate to a method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one insecticidal polynucleotide comprising at least one silencing element, wherein the at least one silencing element is essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, and wherein ingestion of the insecticidal polynucleotide by the insect results in mortality or stunting in the insect. In some embodiments, the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vps l6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S 14, and 60S ribosomal protein L13. In some embodiments, the target gene sequence is selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide comprises a sequence of about 95% to about 100% identity or complementarity to at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975. In some embodiments, the insecticidal polynucleotide is provided in the diet of the insect in the form of a plant or bacterial cell containing the polynucleotide, or as a synthetic polynucleotide molecule, or as a fermentation product (e. g. , a hairpin form of a dsRNA, produced in a bacterium). In some embodiments, the insecticidal polynucleotide is a single -stranded RNA molecule. In some embodiments, the insecticidal polynucleotide is a double- stranded RNA molecule. In some embodiments, the insecticidal polynucleotide is a single -stranded DNA molecule. In some embodiments, the insecticidal polynucleotide is a double-stranded DNA molecule. In some embodiments, the insecticidal polynucleotide is a RNA/DNA hybrid molecule.
[0007] Several embodiments relate to an insecticidal composition comprising an insecticidally effective amount of a polynucleotide, wherein the polynucleotide comprises 18 or more contiguous nucleotides with about 95% to about 100% complementarity with a corresponding portion of a target gene of an insect that infests a plant. In some embodiments, the polynucleotide comprises 21 contiguous nucleotides with a sequence of 100% complementarity with a corresponding portion of the target gene. In some embodiments, the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI
(coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL 14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vps l6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S 14, and 60S ribosomal protein L13. In some embodiments, the polynucleotide comprises 18 or more contiguous nucleotides with about 95% to about 100% complementarity with a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859 or a fragment thereof. In some embodiments, the polynucleotide comprises 21 contiguous nucleotides with a sequence of 100% complementarity with a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859 or a fragment thereof. In some embodiments, the polynucleotide is a single-stranded RNA molecule. In some embodiments, the polynucleotide is molecule is a dsRNA molecule. In some embodiments, the polynucleotide is a single- stranded DNA molecule. In some embodiments, the polynucleotide is a double-stranded DNA molecule. In some embodiments, the polynucleotide is a RNA/DNA hybrid molecule. In some embodiments, the polynucleotide comprises at least one segment (e. g. , an RNA strand or segment of an RNA strand) with a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, polynucleotide comprises at least one segment (e. g. , an RNA strand or segment of an RNA strand) with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, polynucleotide comprises at least one segment of 21 contiguous nucleotides (e. g. , an RNA strand or segment of an RNA strand) that is complementary or identical to a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal composition further comprises one or more of a carrier agent, a surfactant, an organosilicone, a cationic lipid, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator. Embodiments of the insecticidal compositions comprise non-polynucleotide insecticides, e. g. , a bacterially produced insecticidal protein.
[0008] Several embodiments relate to a method of providing a plant having improved resistance to an insect, comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA construct encodes an insecticidal polynucleotide comprising a sequence that is essentially identical or essentially complementary to a fragment of at least one target gene of the insect, and wherein ingestion of the insecticidal polynucleotide by the insect results in mortality or stunting in the insect. In some embodiments, the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In some embodiments, the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide is single -stranded RNA (ssRNA). In other embodiments, the insecticidal polynucleotide is double- stranded RNA (dsR A), which may comprise single-stranded portions, such as a loop in a stem-loop structure. In some embodiments, the insecticidal polynucleotide is a single -stranded DNA molecule. In some embodiments, the insecticidal polynucleotide is a double -stranded DNA molecule. In some embodiments, the insecticidal polynucleotide is a RNA/DNA hybrid molecule. In some embodiments, the insecticidal polynucleotide comprises a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide comprises a sequence of at least 21 contiguous nucleotides that are complementarity or identical to a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975. In some
embodiments, the insecticidal polynucleotide is an RNA (e. g. , an RNA strand or segment of an RNA strand) comprising at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof
[0009] Several embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an insecticidal polynucleotide comprising a sequence of about 95% to about 100% identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. Several embodiments relate to a commercial unit of seed, such as a bag, in which all or substantially all of the seeds comprise a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an insecticidal polynucleotide comprising a sequence of about 95% to about 100% identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide is single-stranded RNA (ssRNA). In other embodiments, the insecticidal polynucleotide is double-stranded RNA
(dsRNA), which may comprise single -stranded portions, such as a loop in a stem-loop structure. In some embodiments, the insecticidal polynucleotide is single -stranded DNA (ssDNA). In other embodiments, the insecticidal polynucleotide is double-stranded DNA (dsDNA), which may comprise single -stranded portions, such as a loop in a stem-loop structure. In some embodiments, the insecticidal polynucleotide is a hybrid RNA/DNA molecule. In some embodiments, the insecticidal polynucleotide comprisesan RNA strand or segment of an RNA strand comprising a sequence of about 95% to about 100% identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide comprises an RNA strand or segment of an RNA strandcomprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0010] In related aspects, provided herein are man-made compositions comprising a insecticidal polynucleotide as described herein, such as ssRNA, dsRNA, ssDNA, dsDNA or hybrid RNA/DNA formulations useful for topical application to a plant or substance in need of protection from an insect infestation; recombinant constructs and vectors useful for making transgenic plant cells and transgenic plants; formulations and coatings useful for treating plants (including plant seeds or propagatable parts such as tubers); plant seeds or propagatable parts such as tubers treated with or containing a polynucleotide as described herein as well as commodity products and foodstuffs produced from such plants; seeds, or propagatable parts (especially commodity products and foodstuffs having a detectable amount of a polynucleotide disclosed herein). Several embodiments relate to polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:l - 859. Several embodiments relate to polyclonal or monoclonal antibodies that bind a peptide or protein encoded by a sequence or a fragment of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or the complement thereof. Such antibodies are made by routine methods as known to one of ordinary skill in the art. DETAILED DESCRIPTION
[0011] The present embodiments relate to methods and compositions for controlling insect pests, in particular the group of coleopteran insects commonly known as "flea beetles", of which there are several genera. Disclosed herein are target genes identified as useful for designing insecticidal polynucleotide agents for preventing or treating flea beetle infestations, especially of commercially important plants. The methods and compositions are especially useful for preventing or treating flea beetle infestations of commercially important Brassica species including species commercially used as oilseed, food, or livestock feed (e. g. , canola, rapeseed, turnips, and field mustard or turnip rape). Such Brassica species include B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B.
septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii, and B. fructiculosa. Also disclosed are sequences for suppressing one or more flea beetle target genes. Several embodiments relate to insecticidal polynucleotide agents that suppress flea beetle target genes. In some embodiments, insecticidal polynucleotides and recombinant DNA molecules and constructs useful in methods of controlling insect pests, especially flea beetles are provided. Several embodiments relate to insecticidal compositions, as well as to transgenic plants resistant to infestation by insect pests. Several embodiments relate to methods of identifying efficacious insecticidal polynucleotide agents, for example, single -stranded RNA molecules, double-stranded RNA molecules, single-stranded DNA molecules, double-stranded DNA molecules, or hybrid RNA/DNA molecules for controlling insect pests and methods for identifying target genes that are likely to represent essential functions, making these genes preferred targets for RNAi -mediated silencing and control of insect pests.
[0012] Several embodiments relate to methods and compositions for inhibiting or controlling flea beetle infestation of a plant by inhibiting in the flea beetle the expression of one or more target gene selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS 14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V- ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In some embodiments, inhibiting the expression of one or more target gene in the flea beetle results in stunting or mortality.
[0013] Several embodiments relate to an insecticidal polynucleotide molecule, such as a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA hybrid, which comprises one or more segments comprising 18 or more contiguous nucleotides, for example 21 or more contiguous nucleotides, having 95% to about 100% (e. g, about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of an insect target gene selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI
(coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S 14, and 60S ribosomal protein LI 3. In some embodiments, the insecticidal polynucleotide comprises multiple segments each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%)
complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide comprises at least 21 contiguous nucleotides having 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide comprises segments complementary to different regions of a target gene, or can comprise multiple copies of a segment. In some embodiments, the insecticidal polynucleotidecomprises multiple segments, each of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a different target gene; in this way multiple target genes, or multiple insect species, can be suppressed.
[0014] Several embodiments relate to a insecticidal polynucleotide (e.g., a ssRNA, a dsRNA, a ssDNA, a dsDNA, or a RNA/DNA hybrid) molecule which inhibits the expression of one or more insect target genes selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. Several embodiments relate to an insecticidal polynucleotide having a length greater than that which is typical of naturally occurring regulatory small R As (such as endogenously produced siRNAs and mature miRNAs), e. g, thepolynucleotide is at least about 30 contiguous base-pairs in length. In some embodiments, the insecticidal polynucleotide has a length of between about 50 to about 500 base-pairs. In some embodiments, the insecticidal polynucleotide is at least 50 base pairs in length. In some embodiments, the insecticidal polynucleotide is formed from two separate, essentially complementary strands (e. g. , where each strand is separately provided, or where each strand is encoded on a separate DNA molecule, or where the two strands are encoded on separate sections of a DNA and are separately transcribed or made separate, for example, by the action of a recombinase or nuclease), wherein at least one strand comprises a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide is double stranded and blunt-ended, e. g. , two separate, equal-length strands which form the double -stranded
polynucleotide through intermolecular hybridisation. In some embodiments, the insecticidal polynucleotide is double stranded and has an overhang at one or both ends (termini), e. g. , two separate, unequal-length strands which form the double -stranded polynucleotide through intermolecular hybridisation; the overhang can be a single nucleotide or 2, 3, 4, 5, 6, or more nucleotides, and can be located on the 5' end or on the 3' end of a strand. In some embodiments, the insecticidal
polynucleotide comprises at least one stem -loop, e. g. , a single polynucleotide molecule that forms a double -stranded region through intramolecular hybridization adjacent to a "hairpin" secondary structure. In some embodiments, the insecticidal polynucleotide is formed from a single self- hybridizing hairpin, wherein one "arm" of the hairpin comprises a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity to at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof In embodiments, self-hybridizing polynucleotides which form hairpins (or partial hairpins) include polynucleotide molecules that comprise "spacer" nucleotides or a single -stranded "loop region" between the double-strand-forming complementary "arms" of sense sequence and anti-sense sequence. In embodiments, such spacers or loops include nucleotides having a sequence unrelated (not complementary or identical to) the target gene corresponding to the double -stranded portion of the hairpin. In embodiments, such spacers or loops include nucleotides having a sequence complementary or identical to the target gene. Examples of spacers or loops include those encoded by SEQ ID NOs:1719 - 1721. In embodiments, the insecticidal polynucleotide comprises multiple stem-loops, with or without spacer nucleotides between each stem-loop. In embodiments, the insecticidal polynucleotide comprises a modified stem-loop such as a "stabilized anti -sense" loop or a "stabilized sense" loop; see, e. g. , United States Patent Numbers 7,855,323 and 9,006,414, which are incorporated by reference in their entirety herein.
[0015] The insecticidal polynucleotide can be chemically synthesized (e. g., by in vitro transcription, such as transcription using a T7 polymerase or other polymerase), or can be produced by expression in a microorganism, by expression in a plant cell, or by microbial fermentation. The insecticidal polynucleotide can be chemically modified, e. g., to improve stability, ease of formulation, or efficacy. In some embodiments, the insecticidal polynucleotide molecule is provided in a microbial or plant cell that expresses the insecticidal polynucleotide (such as a hairpin form of a dsRNA), or in a microbial fermentation product.
[0016] A variety of methods for designing and producing a variety of forms of insecticidal polynucleotide are known in the art and are useful in the compositions and methods disclosed herein. See, for example, the following patents which are incorporated by reference in their entirety herein: ( 1) United States Patent Number 8,598,332 to Waterhouse et al , which discloses recombinant DNA constructs comprising DNA encoding sense RNA and anti-sense RNA sequences in a single transcript that forms an artificial "hairpin" RNA structure with a double-stranded RNA stem by base-pairing between the sense and anti-sense nucleotide sequences; embodiments include hairpins with spacer nucleotides between the sense and anti -sense nucleotide sequences; (2) United States Patent Number 8, 158,414 to Rommens et al , which discloses recombinant DNA constructs including convergently oriented first and second promoters, which produce, e. g. , an RNA duplex that is formed by annealing of two separate RNA transcripts; and (3) United States Patent Numbers 7,855,323 and 9,006,414 to Huang et al , which disclose recombinant DNA constructs including DNA encoding "stabilized anti- sense" transcripts which form a loop of anti -sense-oriented RNA for suppressing one or more target genes; recombinant DNA constructs can be designed to similarly encode "stabilized sense" transcripts which form a loop of sense-oriented RNA for suppressing one or more target genes.
[0017] Embodiments of the compositions comprising insecticical polynucleotides described herein further comprise one or more additional components or adjuvants, e. g. , a carrier agent, an encapsulation agent, an emulsifying agent, a surfactant, an organosilicone, a cationic lipid, a spreading agent, a photoprotective agent, a rainfastness agent, a polynucleotide herbicidal molecule, a non- polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a non-polynucleotide fungicide, a safener, a bait, an insect attractant, an insect pheromone, and an insect growth regulator. In embodiments, these additional components or adjuvants are edible or digestible if ingested by a flea beetle.
[0018] In embodiments, the insecticidal polynucleotide disclosed herein are used in combination with a non-nucleotide pesticidal agent such as a small-molecule pesticidal agent or a proteinaceous pesticidal agent, either concurrently or sequentially. Examples of non-nucleotide pesticidal agents include patatins, plant lectins, phytoecdysteroids, and bacterial insecticidal proteins (e. g. , insecticidal proteins from Bacillus thuringiensis, Xenorhabdus sp., Photorhabdus sp., Brevibacillus laterosporus (previously Bacillus later osporus), Lysinibacillus sphaericus (previously Bacillus sphaericus), Chromobacterium sp., Chromobacterium subtsugae, Paenibacillus sp., Paenibacillus lentimorbus , and Paenibacillus popilliae), a bacterium that produces an insecticidal protein, and an entomicidal bacterial species. In embodiments, the compositions comprising polynucleotides for flea beetle control such as the insecticidal polynucleotide described herein can further comprise, or can be used concurrently or sequentially with, conventional pesticides such as Spiromesifen, Spirodiclofen, Spirotetramat, Pyridaben, Tebufenpyrad, Tolfenpyrad, Fenpyroximate, Flufenerim, Pyrimidifen, Fenazaquin, Rotenone, Cyenopyrafen, Hydramethylnon, Acequinocyl, Fluacrypyrim, Aluminium phosphide, Calcium phosphide, Phosphine, Zinc phosphide, Cyanide, Diafenthiuron, Azocyclotin, Cyhexatin, Fenbutatin oxide, Propargite, Tetradifon, Bensultap, Thiocyclam, Thiosultap-sodium, Flonicamid, Etoxazole, Clofentezine, Diflovidazin, Hexythiazox, Chlorfluazuron, Bistrifluron,
Diflubenzuron, Flucycloxuron, Flufenoxuron, Hexaflumuron, Lufenuron, Novaluron, Noviflumuron, Teflubenzuron, Triflumuron, Buprofezin, Cyromazine, Hydroprene, Kinoprene, Methoprene,
Fenoxycarb, Pyriproxyfen, Pymetrozine, Pyrifluquinazon, Chlorfenapyr, Tralopyril, B. t. {Bacillus thuringiensis) var. aizawai, B.t. var. israelensis, B.t. var. kurstaki, B.t. var. sphaericus, B.t. var.
tenebrionensis, Bacillus thuringiensis crop proteins including CrylAb, CrylAc, CrylFa, Cry2Ab, mCry3A, Cry3Ab, Cry3Bb, Cry34/35Abl, Methyl bromide and other alkyl halides, Chloropicrin, Sulfuryl fluoride, Benclothiaz, Chinomethionat, Cryolite, Methylneodecanamide, Benzoximate, Cymiazole, Fluensulfone, Azadirachtin, Bifenazate, Amidoflumet, Dicofol, Plifenate, Cyflumetofen, Pyridalyl, Beauveria bassiana GHA, Sulfoxaflor, , Spinetoram, Spinosad, Spinosad, Emamectin benzoate, Lepimectin, Milbemectin, Abamectin, Methoxyfenozide, Chromafenozide, Halofenozide, Tebufenozide, Amitraz, Chlorantraniliprole, Cyantraniliprole, Flubendiamide, alpha-endosulfan, Chlordane, Endosulfan, Fipronil, Acetoprole, Ethiprole, Pyrafluprole, Pyriprole, Indoxacarb and Metaflumizone, Acrinathrin, Allethrin, Allethrin-cis-trans, Allethrin-trans, beta-Cyfluthrin, beta- Cypermethrin, Bifenthrin, Bioallethrin, Bioallethrin S-cyclopentenyl, Bioresmethrin, Cycloprothrin, Cyfluthrin, Cyhalothrin, Cypermethrin, Cyphenothrin [(7i?)-trans- isomers], Dimefluthrin, Empenthrin [(EZ)-(IR)- isomers], Esfenvalerate, Etofenprox, Fenpropathrin, Fenvalerate, Flucythrinate, Flumethrin, Gamma-cyhalothryn, lambda-Cyhalothrin, Meperfluthrin, Metofluthrin, Permethrin, Phenothrin [(IR)- trans- isomer], Prallethrin, Profluthrin, Protrifenbute, Resmethrin, Silafluofen, tau-Fluvalinate, Tefluthrin, Tetramethrin, Tetramethrin [(IR)- isomers], Tetramethylfluthrin, theta-Cypermethrin, Tralomethrin, Transfluthrin, zeta-Cypermethrin, alpha-Cypermethrin, Deltamethrin, DDT, and
Methoxychlor, Thiodicarb, Alanycarb, Aldicarb, Bendiocarb, Benfuracarb, Butoxycarboxim, Carbaryl, Carbofuran, Carbosulfan, Ethiofencarb, Fenobucarb, Formetanate, Furathiocarb, Isoprocarb,
Methiocarb, Methomyl, Metolcarb, Oxamyl, Pirimicarb, Propoxur, Thiofanox, Triazamate,
Trimethacarb, XMC, Xylylcarb, Chlorpyrifos, Malathion, Acephate, Azamethiphos, Azinphos-ethyl, Azinphos-methyl, Cadusafos, Chlorethoxyfos, Chlorfenvinphos, Chlormephos, Chlo yrifos-methyl, Coumaphos, Cyanophos, Demeton-S-methyl, Diazinon, Dichlorvos/DDVP, Dicrotophos, Dimethoate, Dimethylvinphos, Disulfoton, EPN, Ethion, Ethoprophos, Famphur, Fenamiphos, Fenitrothion, Fenthion, Fonofos, Fosthiazate, Imicyafos, Isofenphos-methyl, Mecarbam, Methamidophos, Methidathion, Mevinphos, Monocrotophos, Naled, Omethoate, Oxydemeton-methyl, Parathion, Parathion-methyl, Phenthoate, Phorate, Phosalone, Phosmet, Phosphamidon, Phoxim, Pirimiphos-ethyl, Profenofos, Propaphos, Propetamphos, Prothiofos, Pyraclofos, Pyridaphenthion, Quinalphos, Sulfotep, Tebupirimfos, Temephos, Terbufos, Tetrachlorvinphos, Thiometon, Triazophos, Trichlorfon,
Vamidothion Imidacloprid, Thiamethoxam, Acetamiprid, Clothianidin, Dinotefuran, Nitenpyram, Nithiozine, Nicotine, Thiacloprid, chlorantraniliprole, and cyantraniliprole. In embodiments, the compositions comprising insecticidal polynucleotide for flea beetle control in Brassica species, including canola, further comprise, or are used concurrently or sequentially with, foliar sprays including one or more pesticides selected from the group consisting of Deltamethrin, Cypermethrin, Lambda- cyhalothrin, Permethrin, Carbaryl, Carbofuran, and Malathion, or seed treatments comprising one or more pesticides selected from the group consisting of Thiamethoxam, Imidacloprid, and Clothianadin.
[0019] In embodiments, the compositions comprising insecticidal polynucleotides for flea beetle control described herein can further comprise, or can be used concurrently or sequentially with, conventional fungicides such as bupirimate, dimethirimol, ethirimol, cyprodinil, pyrimethanil, mepanipyrim, fenpiclonil, fludioxonil; phenylamides, benalaxyl, furalaxyl, metalaxyl, i?-metalaxyl, ofurace, oxadixyl, benomyl, carbendazim, debacarb, fuberidazole, thiabendazole, chlozolinate, dichlozoline, iprodine, myclozoline, procymidone, vinclozolin, carboxin, fenfuram, flutolanil, mepronil, oxycarboxin, thifluzamide, guazatine, dodine, iminoctadine, azoxystrobin, kresoxim-methyl, metominostrobin, or trifloxystrobin, ferbam, mancozeb, maneb, metiram, propineb, thiram, zineb, ziram, captafol, captan, dichlofluanid, fluoromide, folpet, tolyfluanid, copper hydroxide, copper oxychloride, copper sulfate, cuprous oxide, mancopper, oxine-copper, dinocap, nitrothal-isopropyl, edifenphos, iprobenphos, isoprothiolane, phosdiphen, pyrazopho, toclofos-methyl, acibenzolar-iS*- methyl, haφin, anilazine, blasticidin-S, chinomethionat, chloroneb, chlorothalonil, cymoxanil, dichione, diclomezine, dicloran, diethofencarb, dimethomorph, dithianon, etridiazole, famoxadone, fenamidone, fentin, fenpyrazamine, ferimzone, fluazinam, flusulfamide, fosetyl-aluminium, hymexazol, kasugamycin, methasulfocarb, pencycuron, phthalide, polyoxins, probenazole, propamocarb, pyroquilon, quinoxyfen, quintozene, sulfur, triazoxide, tricyclazole, validamycin, azaconazole, bitertanol, propiconazole, difenoconazole, diniconazole, cyproconazole, epoxiconazole,
fluquinconazole, flusilazole, flutriafol, hexaconazole, imazalil, imibenconazole, ipconazole, tebuconazole, tetraconazole, fenbuconazole, metconazole, myclobutanil, perfurazoate, penconazole, bromuconazole, pyrifenox, prochloraz, triadimefon, triadimenol, triflumizol, triticonazole, triforine, ancymidol, fenarimol or nuarimol, dodemorph, fenpropidin, fenpropimoφh, spiroxamine, tridemoφh, and fenhexamid. In embodiments, the compositions comprising insecticidal polynucleotides for flea beetle control in Brassica species, including canola, further comprise, or are used concurrently or sequentially with, foliar sprays including one or more fungicides selected from the group consisting of Azoxystrobin, Bacillus subtilis strain QST 2808, Boscalid, Fluxopyroxad, Pyraclostrobin,
Metconazole, Prothioconazole, Penthiopyrad, Picoxystrobin, and Thiophanate Methyl, or seed treatments including one or more fungicides selected from the group consisting of Azoxystrobin, Metalaxyl, Trifloxystrobin, Pyraclostrobin, Sedaxane, Penflufen, Fludioxonil, and Mefenoxam.
[0020] In embodiments, the compositions comprising insecticidal polynucleotides for flea beetle described herein can further comprise, or can be used concurrently or sequentially with, conventional herbicides such as glyphosate, auxin-like herbicides such as dicamba, phosphinothricin, glufosinate, 2,2-dichloropropionic acid (Dalapon), acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates, and phthalide, bromoxynil,
cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop), sulfonamide herbicides, triazine herbicides, 5-methyltryptophan, aminoethyl cysteine, pyridazinone herbicides such as norflurazon, cyclopropylisoxazole herbicides such as isoxaflutole, protoporphyrinogen oxidase inhibitors, herbicidea containing an aryloxyalkanoate moiety, phenoxy auxins such as 2,4-D and dichlorprop, pyridyloxy auxins such as fluroxypyr and triclopyr, aryloxyphenoxypropionate s (AOPP) acetyl- coenzyme A carboxylase (ACCase) inhibitors such as haloxyfop, quizalofop, and diclofop, and 5- substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors such as pyraflufen and flumiclorac. In embodiments, the compositions comprising insecticidal polynucleotides for flea beetle control in Brassica species, including canola, further comprise, or are used concurrently or sequentially with, one or more post-emergence herbicides selected from the group consisting of Quizalofop, Sethoxydim, Clethodim, and Clopyralid. In embodiments, the compositions comprising insecticidal polynucleotides for flea beetle control in herbicide-resistant Brassica species, including herbicide- resistant canola, further comprise, or are used concurrently or sequentially with, one or more herbicides selected from the group consisting of Imazamox, Glyphosate, and Glufosinate.
[0021] The compositions and methods disclosed are useful for inhibiting or controlling flea beetle infestation of a plant, such as a. Brassica species. In embodiments, the compositions and methods are used to treat a growing plant, such as a field of Brassica plants. Embodiments include compositions comprising insecticidal polynucleotides disclosed herein in a composition in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment composition. In embodiments, such compositions are applied to a surface of the plant in need of protection from or treatment for flea beetle infestations, or applied directly to the flea beetles, or provided in an ingestible form to the flea beetles. In embodiments, a composition comprising insecticidal polynucleotides disclosed herein is applied directly to
ungerminated seeds (such as ungerminated Brassica species seeds), providing plants germinated from the treated seeds increased resistance to flea beetle infestations; examples of seed treatment methods are disclosed in United States Patent Application 14/143,836, which is incorporated by reference in its entirety herein. An embodiment includes a Brassica seed that is treated by directly contacting the seed with an insecticidal polynucleotide (such as a ssRNA, dsRNA, ssDNA, dsDNA or R A/DNA hybrid molecule) disclosed herein, followed by germination into a Brassica plant that exhibits increased resistance to flea beetle infestations. [0022] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used and the manufacturing or laboratory procedures described below are well known and commonly employed in the art. Conventional methods are used for these procedures, such as those provided in the art and various general references. Where a term is provided in the singular, the inventors also contemplate aspects described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, "The American
Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton
Mifflin Harcourt, Boston and New York), the "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York), or the "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.
[0023] Unless otherwise stated, nucleic acid sequences in the text of this specification are given, when read from left to right, in the 5' to 3' direction. One of skill in the art would be aware that a given DNA sequence is understood to define a corresponding RNA sequence which is identical to the DNA sequence except for replacement of the thymine (T) nucleotides of the DNA with uracil (U) nucleotides. Thus, providing a specific DNA sequence is understood to define the RNA equivalent and vice versa. A given first polynucleotide sequence, whether DNA or RNA, further defines the sequence of its exact complement (which can be DNA or RNA), /'. e. , a second polynucleotide that hybridizes perfectly to the first polynucleotide by forming Watson-Crick base-pairs. By "essentially
complementary" is meant that a polynucleotide strand (or at least one strand of a double-stranded polynucleotide) is designed to hybridize (generally under physiological conditions such as those found in a living plant or animal cell) to a target gene or to a fragment of a target gene or to the transcript of the target gene or the fragment of a target gene; one of skill in the art would understand that such hybridization does not necessarily require 100% sequence complementarity. A first nucleic acid sequence is "operably" connected or "linked" with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter sequence is "operably linked" to DNA if the promoter provides for transcription or expression of the DNA. Generally, operably linked DNA sequences are contiguous.
[0024] The term "polynucleotide" commonly refers to a DNA or RNA molecule containing multiple nucleotides and generally refers both to "oligonucleotides" (a polynucleotide molecule of 18- 25 nucleotides in length) and longer polynucleotides of 26 or more nucleotides. Polynucleotides also include molecules containing multiple nucleotides, including non-canonical nucleotides or chemically modified nucleotides as commonly practiced in the art; see, e. g, chemical modifications disclosed in the technical manual "RNA Interference (RNAi) and DsiRNAs", 2011 (Integrated DNA Technologies Coralville, IA). [0025] Generally, insecticidal polynucleotides as described herein, whether DNA or RNA or both, and whether single- or double -stranded, comprise at least one segment of 18 or more contiguous nucleotides (or, in the case of double-stranded polynucleotides, at least 18 contiguous base-pairs) that are essentially identical or complementary to a fragment of equivalent size of the DNA of a target gene or the target gene's RNA transcript. Throughout this disclosure, "at least 18 contiguous" means "from about 18 to about 10,000, including every whole number point in between" . Thus, embodiments include compositions comprising oligonucleotides having a length of 18-25 nucleotides (18-mers, 19- mers, 20-mers, 21-mers, 22-mers, 23-mers, 24-mers, or 25-mers), or medium-length polynucleotides having a length of 26 or more nucleotides (polynucleotides of 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, about 65, about 70, about 75, about 80, about 85, about 90, about 95, about 100, about 1 10, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, or about 300
nucleotides), or long polynucleotides having a length greater than about 300 nucleotides (e. g , polynucleotides of between about 300 to about 400 nucleotides, between about 400 to about 500 nucleotides, between about 500 to about 600 nucleotides, between about 600 to about 700 nucleotides, between about 700 to about 800 nucleotides, between about 800 to about 900 nucleotides, between about 900 to about 1000 nucleotides, between about 300 to about 500 nucleotides, between about 300 to about 600 nucleotides, between about 300 to about 700 nucleotides, between about 300 to about 800 nucleotides, between about 300 to about 900 nucleotides, or about 1000 nucleotides in length, or even greater than about 1000 nucleotides in length, for example up to the entire length of a target gene including coding or non-coding or both coding and non-coding portions of the target gene). Where an insecticidal polynucleotide is double -stranded, such as the dsRNAs described in the working Examples, its length can be similarly described in terms of base pairs. Double -stranded insecticidal
polynucleotides can further be described in terms of one or more of the single -stranded components.
[0026] The insecticidal polynucleotides described herein can be single-stranded (ss) or double -stranded (ds). "Double-stranded" refers to the base-pairing that occurs between sufficiently complementary, anti-parallel nucleic acid strands to form a double -stranded nucleic acid structure, generally under physiologically relevant conditions. Embodiments include those wherein the polynucleotide is selected from the group consisting of sense single -stranded DNA (ssDNA), sense single -stranded RNA (ssRNA), double-stranded RNA (dsRNA), double -stranded DNA (dsDNA), a double -stranded DNA/RNA hybrid, anti-sense ssDNA, or anti-sense ssRNA; a mixture of
polynucleotides of any of these types can be used. In some embodiments, the insecticidal
polynucleotide is double-stranded RNA of a length greater than that which is typical of naturally occurring regulatory small RNAs (such as endogenously produced siRNAs and mature miRNAs). In some embodiments, the insecticidal polynucleotide is double-stranded RNA of at least about 30 contiguous base-pairs in length. In some embodiments, the insecticidal polynucleotide is double- stranded RNA with a length of between about 50 to about 500 base-pairs. In some embodiments, the insecticidal polynucleotide can comprise components other than standard ribonucleotides, e. g., an embodiment is an RNA that comprises terminal deoxyribonucleotides.
[0027] Insecticidal polynucleotides of any size can be used, alone or in combination, in the various methods and compositions described herein. In some embodiments, a single insecticidal polynucleotide is used to make a composition (e. g., a composition for topical application). In other embodiments, a mixture or pool of different insecticidal polynucleotides is used; in such cases the insecticidal polynucleotides can be for a single target gene or for multiple target genes.
[0028] In various embodiments, an insecticidal polynucleotide as described herein comprises naturally occurring nucleotides, such as those which occur in DNA and RNA. In certain embodiments, the insecticidal polynucleotide is a combination of ribonucleotides and deoxyribonucleotides, for example, synthetic polynucleotides consisting mainly of ribonucleotides but with one or more terminal deoxyribonucleotides or one or more terminal dideoxyribonucleotides or synthetic polynucleotides consisting mainly of deoxyribonucleotides but with one or more terminal dideoxyribonucleotides. In certain embodiments, the insecticidal polynucleotide comprises non-canonical nucleotides such as inosine, thiouridine, or pseudouridine. In certain embodiments, the insecticidal polynucleotide comprises chemically modified nucleotides. Examples of chemically modified oligonucleotides or polynucleotides are well known in the art; see, for example, U.S. Patent Publication 2011/0171287, U.S. Patent Publication 2011/0171176, U.S. Patent Publication 2011/0152353, U.S. Patent Publication 2011/0152346, and U.S. Patent Publication 2011/0160082, which are herein incorporated by reference. Illustrative examples include, but are not limited to, the naturally occurring phosphodiester backbone of an oligonucleotide or polynucleotide which can be partially or completely modified with
phosphorothioate, phosphorodithioate, or methylphosphonate internucleotide linkage modifications, modified nucleoside bases or modified sugars can be used in oligonucleotide or polynucleotide synthesis, and oligonucleotides or polynucleotides can be labeled with a fluorescent moiety (e. g., fluorescein or rhodamine) or other label (e. g., biotin).
[0029] The term "recombinant", as used to refer to a polynucleotide (such as insecticidal polynucleotide molecules or recombinant DNA constructs described herein), means that the polynucleotide is not a naturally occurring molecule, /'. e. , that human intervention is required for the polynucleotide to exist. A recombinant polynucleotide is produced using recombinant nucleic acid techniques, or by chemical synthesis, and can include combinations of sequences that do not occur in nature (e. g. , combinations of a heterologous promoter and a DNA encoding an RNA to be expressed, or an RNA molecule that comprises concatenated segments of a target gene that do not in nature occur adjacent to one another). A recombinant polynucleotide can be biologically produced in a cell (such as a bacterial or plant or animal cell), for example, when that cell is transfected or transformed with a vector encoding the recombinant polynucleotide (e. g. , a vector encoding a hairpin form of a dsRNA, produced in a bacterium). A recombinant polynucleotide can comprise sequences of nucleotides designed in silico using appropriate algorithms. [0030] The insecticidal polynucleotides disclosed herein are generally designed to suppress or silence one or more genes ("target genes"). The term "gene" refers to any portion of a nucleic acid that provides for expression of a transcript or encodes a transcript, or that is a hereditable nucleic acid sequence. A "gene" can include, but is not limited to, a promoter region, 5 ' untranslated regions, transcript encoding regions that can include intronic regions, 3 ' untranslated regions, or combinations of these regions. In embodiments, the target genes can include coding or non-coding sequence or both. In other embodiments, the target gene has a sequence identical to or complementary to a messenger RNA, e. g. , in embodiments the target gene is a cDNA.
[0031] As used herein, the term "isolated" refers to separating a molecule from other molecules normally associated with it in its native or natural state. The term "isolated" thus may refer to a DNA molecule that has been separated from other DNA molecule(s) which normally are associated with it in its native or natural state. Such a DNA molecule may be present in a recombined state, such as a recombinant DNA molecule. Thus, DNA molecules fused to regulatory or coding sequences with which they are not normally associated, for example as the result of recombinant techniques, are considered isolated, even when integrated as a transgene into the chromosome of a cell or present with other DNA molecules.
[0032] By "insecticidally effective" is meant effective in inducing a physiological or behavioural change in an insect (e. g. , adult or larval flea beetles) that infests a plant such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity or decreased fecundity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. In some embodiments, application of an insecticidally effective amount of the insecticidal polynucleotide, such as a dsRNA molecule, to a plant improves the plant's resistance to infestation by the insect. In some embodiments, application of an insecticidally effective amount of the insecticidal polynucleotide to a crop plant improves yield (e. g. , increased biomass, increased seed or fruit production, or increased oil, starch, sugar, or protein content) of that crop plant, in comparison to a crop plant not treated with the insecticidal polynucleotide. While there is no upper limit on the concentrations and dosages of an insecticidal polynucleotide as described herein that can be useful in the methods and compositions provided herein, lower effective concentrations and dosages will generally be sought for efficiency and economy. Non-limiting embodiments of effective amounts of an insecticidal polynucleotide include a range from about 10 nanograms per milliliter to about 100 micrograms per milliliter of an insecticidal polynucleotide in a liquid form sprayed on a plant, or from about 10 milligrams per acre to about 100 grams per acre of polynucleotide applied to a field of plants, or from about 0.001 to about 0.1 microgram per milliliter of polynucleotide in an artificial diet for feeding the insect. Where compositions as described herein are topically applied to a plant, the concentrations can be adjusted in consideration of the volume of spray or treatment applied to plant leaves or other plant part surfaces, such as flower petals, stems, tubers, fruit, anthers, pollen, leaves, roots, or seeds. In one embodiment, a useful treatment for herbaceous plants using insecticidal polynucleotides as described herein is about 1 nanomole (nmol) of polynucleotides per plant, for example, from about 0.05 to 1 nmol polynucleotides per plant. Other embodiments for herbaceous plants include useful ranges of about 0.05 to about 100 nmol, or about 0.1 to about 20 nmol, or about 1 nmol to about 10 nmol of polynucleotides per plant. In certain embodiments, about 40 to about 50 nmol of a single-stranded polynucleotide as described herein are applied. In certain embodiments, about 0.5 nmol to about 2 nmol of an insecticidal polynucleotide as described herein is applied. In certain embodiments, a composition containing about 0.5 to about 2.0 milligrams per milliliter, or about 0.14 milligrams per milliliter of an insecticidal polynucleotide (or a single-stranded 21-mer) as described herein is applied. In certain embodiments, a composition of about 0.5 to about 1.5 milligrams per milliliter of an insecticidal polynucleotide as described herein of about 50 to about 200 or more nucleotides is applied. In certain embodiments, about 1 nmol to about 5 nmol of an insecticidal polynucleotide as described herein is applied to a plant. In certain embodiments, a polynucleotide composition as topically applied to the plant comprises at least one polynucleotide as described herein at a concentration of about 0.01 to about 10 milligrams per milliliter, or about 0.05 to about 2 milligrams per milliliter, or about 0.1 to about 2 milligrams per milliliter. Very large plants, trees, or vines can require correspondingly larger amounts of insecticidal polynucleotides. When using long insecticidal polynucleotide molecules that can be processed into multiple oligonucleotides (e. g. , multiple oligonucleotides encoded by a single recombinant DNA molecule as disclosed herein), lower concentrations can be used. Non-limiting examples of effective insecticidal polynucleotide treatment regimes include a treatment of between about 0.1 to about 1 nmol of polynucleotide molecule per plant, or between about 1 nmol to about 10 nmol of polynucleotide molecule per plant, or between about 10 nmol to about 100 nmol of polynucleotide molecule per plant.
METHODS OF CAUSING INSECT MORTALITY AND OF CONTROLLING INSECT INFESTATIONS
[0033] Several embodiments relate to a method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one insecticidal polynucleotide comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of the insect, wherein the target gene sequence is selected from the group consisting of SEQ ID NOs:l - 859, and wherein ingestion of the insecticidal polynucleotide by the insect results in mortality or stunting in the insect. These methods are useful for controlling insect infestations of a plant, for example for prevention or treatment of a flea beetle infestation of a crop plant, particularly commercially important Brassica species.
[0034] In embodiments, the at least one silencing element comprises a sequence having about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In embodiments, the at least one silencing element comprises 18 or more contiguous nucleotides with a sequence of 100%
complementarity with a fragment of the target gene of the insect, wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In embodiments, the at least one silencing element comprises at least one segment of 18 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In embodiments, the at least one silencing element comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In embodiments the at least one silencing element comprises a sequence of about 95% to about 100% identity or
complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0035] In embodiments, the insecticidal polynucleotide is provided in a microbial or plant cell that expresses the insecticidal polynucleotide, or in a microbial fermentation product, or is chemically synthesized. In embodiments, the insecticidal polynucleotide comprises dsRNA. In embodiments, the dsRNA is blunt-ended, or has an overhang at at least one terminus, or comprises at least one stem -loop. In embodiments, the dsRNA is provided by convenient techniques commonly used. In embodiments, the dsRNA is chemically synthesized, produced by expression in a
microorganism, produced by expression in a plant cell, or produced by microbial fermentation. In embodiments, the dsRNA is made from naturally occurring ribonucleotides; in other embodiments the dsRNA is chemically modified.
[0036] In embodiments, the method is useful for causing mortality or stunting in insects that are pests of commercially important crop plants, such as an insect pest of a Brassica species. In embodiments, the insect is a flea beetle. In embodiments, the insect is a species of a genus selected from the group consisting of the genera. Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus , Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona,
Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola,
Sinaltica, Sphaeroderma, Systena, Trachy aphthona, Xuthea, and Zipangia. In embodiments, the insect is a species selected from the group consisting oiAltica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle). In embodiments, the insect is a species selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes punctulata (hop flea beetle).
[0037] Embodiments of the method include those in which the insecticidal polynucleotide is designed to silence a target gene in a genus- or species-specific manner, for example, wherein (a) the insect is a Phyllotreta species and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 551; (b) the insect is Phyllotreta atra (turnip flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296; (c) the insect is Phyllotreta cruciferae (canola flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532; (d) the insect is Phyllotreta striolata (striped flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551; (e) the insect is a Psylliodes species and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859; or (f) the insect is Psylliodes chrysocephala and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859. Embodiments of the method also include those wherein (a) the insect is a. Phyllotreta species and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1410 or a fragment thereof; (b) the insect is Phyllotreta atra (turnip flea beetle) and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof; (c) the insect is Phyllotreta cruciferae (canola flea beetle) and insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731-1972, and 1974 or a fragment thereof; (d) the insect is Phyllotreta striolata (striped flea beetle) and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975 or a fragment thereof; (e) the insect is a Psylliodes species and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1411 - 1718 or a fragment thereof; or (f) the insect is Psylliodes chrysocephala and the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1411 - 1718 or a fragment thereof.
[0038] Embodiments of the method include those wherein at least one insecticidal polynucleotide is provided in a composition comprising the insecticidal polynucleotide , wherein the composition is applied to a surface of the insect or to a surface of a seed or plant in need of protection from infestation by the insect. Embodiments of such compositions include those where the
composition is a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment. In many embodiments, the composition is formulated in a form that is ingested by the insect. In embodiments, the composition further includes one or more components selected from the group consisting of a carrier agent, a surfactant, an organo silicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non- polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator. In embodiments, the composition further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
Paenibacillus popilliae.
[0039] Several embodiments relate to a method for controlling an insect infestation of a plant comprising contacting the plant and/or an insect that infests a plant with a insecticidal polynucleotide, wherein the insecticidal polynucleotide comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of a target gene of the insect selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta,
RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain,
ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V- ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In some embodiments, the insecticidal polynucleotide comprises at least one segment of 18 or more contiguous nucleotides with a sequence of about 95% to about 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the insecticidal polynucleotide comprises at least one segment of 21 contiguous nucleotides with a sequence of 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In this context "controlling" includes inducement of a physiological or behavioural change in an insect (adult or larvae or nymphs) such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. In some embodiments, the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof, or the complement thereof.
[0040] In various embodiments, the insect is a flea beetle, e. g., a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea,
Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera,
Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus , Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona,
Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachy aphthona, Xuthea, and Zipangia. In some embodiments, the insect is selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle). In some embodiments, the insect is selected from the group consisting of Phyllotreta armoraciae
(horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle),
Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes punctulata (hop flea beetle).
[0041] The plant can be any plant that is subject to infestation by an insect that can be controlled by the insecticidal polynucleotides disclosed herein. Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants". The method is especially useful for controlling an insect infestation of an ornamental plant or a crop plant. Various embodiments of the method include those wherein the plant is a plant in the family Brassicaceae, including a Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B.
tournefortii , and B. fructiculosa. In other embodiments, the plant is selected from the group consisting of Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
[0042] Methods include those developed for specific flea beetle pests for a given plant, e. g. , wherein the plant is a potato plant and the insect is Epitrix cucumeris (potato flea beetle). In some embodiments, specific target genes are identified as targets for RNAi-mediated control in a given insect species. Various embodiments of the method include those wherein (a) the insect is Phyllotreta atra (turnip flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296; (b) the insect is Phyllotreta cruciferae (canola flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532; (c) the insect is Phyllotreta striolata (striped flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551; or (d) the insect is Psylliodes chrysocephala and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859.
[0043] In some embodiments, specific insecticidal polynucleotides are developed for specific target genes in a given insect species. Embodiments of the method include those wherein (a) the insect is Phyllotreta atra (turnip flea beetle) and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof; (b) the insect is Phyllotreta cruciferae (canola flea beetle) and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731-1972, and 1974 or a fragment thereof; (c) the insect is Phyllotreta striolata (striped flea beetle) and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975 or a fragment thereof; or (d) the insect is Psylliodes
chrysocephala and the insecticidal polynucleotide comprises at least one sequence selected from the group consisting of SEQ ID NOs:1411 - 1718 or a fragment thereof.
[0044] The method includes contacting an insect, such as a flea beetle, with an insecticidal polynucleotide . Embodiments include contacting via oral delivery to the insect, or non-oral delivery to the insect, or a combination of oral and non-oral delivery to the insect. Embodiments include contacting insects in the adult stage, or in larval stages, or in the egg stage. In some embodiments, contacting results in mortality (death) or stunting (growth stunting or decrease in or cessation of metamorphosis stage development) of the insect, thereby preventing or treating infestation of the plant by the insect. In some embodiments, contacting results in inducement of a physiological or behavioural change in an insect (adult or larvae or nymphs) that results in a decreased ability of the insect to infest or damage a plant, for example, a decrease in reproductive capacity, or a decrease in or cessation of feeding behavior or movement.
[0045] In some embodiments of the method, the contacting comprises application of a composition comprising one or more insecticidal polynucleotide to a surface of the insect or to a surface of the plant infested by the insect. The composition can comprise or be in the form of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment. In some embodiments, the contacting comprises providing the insecticidal polynucleotide in a composition that further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator. In some embodiments, the contacting comprises providing the one or more insecticidal polynucleotides in a composition that further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus {Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus , and Paenibacillus popilliae.
[0046] In some embodiments of the method, the contacting comprises providing one or more insecticidal polynucleotides in a composition that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. Such compositions can further comprise one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non- polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator. Such compositions can further comprise at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species,
Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus {Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus , and Paenibacillus popilliae. In embodiments, the combination of the insecticidal polynucleotide and the non-polynucleotide pesticidal agent provides a level of insect control that is greater than the sum of the effects of the insecticidal polynucleotide and the non-polynucleotide pesticidal agent components if tested separately.
INSECTICIDAL COMPOSITIONS
[0047] Several embodiments relate to an insecticidal composition comprising an
insecticidally effective amount of an insecticidal polynucleotide, such as a ssR A, dsR A, ssDNA, dsDNA or hybrid RNA/DNA molecule, wherein the insecticidal polynucleotide comprises at least 18 or more contiguous nucleotides with a sequence of about 95% to about 100% {e. g. , about 95%, about
96%, about 97%, about 98%, about 99%, or about 100%) complementarity with a fragment of an insect target gene selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In some embodiments the insecticidal polynucleotide comprises at least 18 or more contiguous nucleotides having about 95% to about 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments the insecticidal polynucleotide comprises at least 21 contiguous nucleotides having 100% complementarity with a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In embodiments, the insecticidal polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0048] In various embodiments of the insecticidal composition, the insect is a flea beetle, e. g., a species of a genus selected from the group consisting of the genemAltica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis,
Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus ,
Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and Zipangia. In some embodiments, the insect is selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle). In some embodiments, the insect is selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes punctulata (hop flea beetle).
[0049] The insecticidal composition is useful for treating a plant or area in the vicinity of a plant to provide protection or treatment from insects, especially flea beetles. A related aspect is a plant treated with an insecticidal composition as described herein, or a seed of the treated plant, wherein the plant exhibits improved resistance to the insect (e. g. , improved resistance to flea beetles). In some embodiments, the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not treated with the insecticidal composition. In an embodiment, yield (oilseed biomass or oil content) in canola or oilseed rape plants is improved by application of an insecticidally effective amount of a insecticidal polynucleotidetargetting one or more genes identified from Phyllotreta cruciferae (canola flea beetle); in particular embodiments, the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532. The plant can be any plant that is subject to infestation by an insect that can be controlled by the insecticidal composition. Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants". The method is especially useful for controlling an insect infestation of an ornamental plant or a crop plant. Various embodiments include those wherein the plant is a plant in the family Brassicaceae, including a Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii, and B. fructiculosa. In other embodiments, the plant is selected from the group consisting of Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
[0050] In some embodiments, the insecticidal composition is developed for specific flea beetle pests for a given plant, e. g. , where the plant is a potato plant and the insect is Epitrix cucumeris (potato flea beetle). In some embodiments, the insecticidal composition is developed for specific target genes in a given insect species. Specific embodiments of the insecticidal composition include those wherein (a) the insect is Phyllotreta atra (turnip flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296; (b) the insect is Phyllotreta cruciferae (canola flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532; (c) the insect is Phyllotreta striolata (striped flea beetle) and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551; or (d) the insect is Psylliodes chrysocephala and the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859
[0051] In some embodiments the insecticidal polynucleotide molecule of use in this method is provided as an isolated insecticidal polynucleotide molecule (not part of an expression construct, e. g. , lacking additional elements such as a promoter or terminator sequences). Such insecticidal polynucleotide molecules can be relatively short, such as single- or double-stranded RNA, DNA or hybrid RNA/DNA molecules of between about 18 to about 300 or between about 50 to about 500 nucleotides (for single -stranded polynucleotides) or between about 18 to about 300 or between about 50 to about 500 base-pairs (for double-stranded polynucleotides). In embodiments the polynucleotide is a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0052] In some embodiments, the insecticidal composition is in a form selected from the group consisting of a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment. In some embodiments, the insecticidal composition is provided in a form that is ingested by the insect, such as in a liquid, emulsion, or powder applied to a plant on which the insect feeds, or in the form of bait. The insecticidal compositions can further comprise one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator. The insecticidal compositions can further comprise at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus , and Paenibacillus popilliae. In some embodiments, the combination of the insecticidal polynucleotide molecule and the non-polynucleotide pesticidal agent provides a level of insect control that is greater than the sum of the effects of the insecticidal polynucleotide molecule and the non- polynucleotide pesticidal agent components if tested separately. [0053] Embodiments of the compositions optionally comprise the appropriate stickers and wetters required for efficient foliar coverage as well as UV protectants to protect insecticidal polynucleotides, such as dsR As, from UV damage. Such additives are commonly used in the bioinsecticide industry and are known to those skilled in the art. Compositions for soil application can comprise granular formulations that serve as bait for insect larvae. Embodiments include a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, an insect attractant, and an insect growth regulator.
[0054] Embodiments of compositions may comprise a "transfer agent", an agent that, when combined with a composition comprising an insecticidal polynucleotide as disclosed herein that is topically applied to the surface of an organism, enables the polynucleotide to enter the cells of that organism. Such transfer agents can be incorporated as part of the composition comprising a insecticidal polynucleotide as disclosed herein, or can be applied prior to, contemporaneously with, or following application of the composition comprising an insecticidal polynucleotide as described herein. In some embodiments, a transfer agent is an agent that improves the uptake of an insecticidal polynucleotide by an insect. In some embodiments, a transfer agent is an agent that conditions the surface of plant tissue, e. g , seeds, leaves, stems, roots, flowers, or fruits, to permeation by an insecticidal polynucleotide into plant cells. In some embodiments, the transfer agent enables a pathway for a polynucleotide through cuticle wax barriers, stomata, and/or cell wall or membrane barriers into plant cells.
[0055] Suitable transfer agents include agents that increase permeability of the exterior of the organism or that increase permeability of cells of the organism to polynucleotides. Suitable transfer agents include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning or transfer include (a) surfactants, (b) an organic solvent or an aqueous solution or aqueous mixtures of organic solvents, (c) oxidizing agents, (d) acids, (e) bases, (f) oils, (g) enzymes, or combinations thereof. In some embodiments, application of an insecticidal polynucleotide and a transfer agent optionally includes one or more of an incubation step, a neutralization step (e. g. , to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. Suitable transfer agents can be in the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition, or can cause the polynucleotide composition to take the form of an emulsion, a reverse emulsion, a liposome, or other micellar-like composition. Embodiments of transfer agents include counter-ions or other molecules that are known to associate with nucleic acid molecules, e. g , cationic lipids, inorganic ammonium ions, alkyl ammonium ions, lithium ions, polyamines such as spermine, spermidine, or putrescine, and other cations. Embodiments of transfer agents include organic solvents such as DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, or other solvents miscible with water or that dissolve
phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Embodiments of transfer agents include naturally derived or synthetic oils with or without surfactants or emulsifiers, e. g. , plant-sourced oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available on-line at herbicide.adjuvants.com), paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N- pyrrolidine.
[0056] Embodiments of transfer agents include organosilicone preparations. For example, a suitable transfer agent is an organosilicone preparation that is commercially available as SILWET L- 77® brand surfactant having CAS Number 27306-78-1 and EPA Number: CAL. REG NO. 5905-50073- AA, and currently available from Momentive Performance Materials, Albany, New York. In embodiments where a SILWET L-77® brand surfactant organosilicone preparation is used as transfer agent in the form of a spray treatment (applied prior to, contemporaneously with, or following application of the composition comprising an insecticidal polynucleotide as disclosed herein) of plant leaves or other plant surfaces, freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g , about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent) are efficacious in preparing a leaf or other plant surface for transfer of a polynucleotide as disclosed herein into plant cells from a topical application on the surface. One embodiment includes a composition that comprises a polynucleotide and a transfer agent including an organosilicone preparation such as Silwet L-77 in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent). One embodiment includes a composition that comprises a polynucleotide and a transfer agent including SILWET L-77® brand surfactant in the range of about 0.3 to about 1 percent by weight (wt percent) or about 0.5 to about \%t by weight (wt percent).
[0057] Organosilicone compounds useful as transfer agents for use in compositions and methods disclosed herein include, but are not limited to, compounds that include: (a) a trisiloxane head group that is covalently linked to, (b) an alkyl linker including, but not limited to, an ^-propyl linker, that is covalently linked to, (c) a polyglycol chain, that is covalently linked to, (d) a terminal group. Trisiloxane head groups of such organosilicone compounds include, but are not limited to,
heptamethyltrisiloxane. Alkyl linkers can include, but are not limited to, an ^-propyl linker. Polyglycol chains include, but are not limited to, polyethylene glycol or polypropylene glycol. Polyglycol chains can include a mixture that provides an average chain length "n" of about "7.5". In certain
embodiments, the average chain length "n" can vary from about 5 to about 14. Terminal groups can include, but are not limited to, alkyl groups such as a methyl group. Organosilicone compounds useful as transfer agents for use in compositions and methods disclosed herein include, but are not limited to, trisiloxane ethoxylate surfactants or polyalkylene oxide modified heptamethyl trisiloxane. An example of a transfer agent for use in compositions and methods disclosed herein is Compound I:
Figure imgf000029_0001
(Compound I: polyalkyleneoxide heptamethyltrisiloxane, average n=7.5).
[0058] Organo silicone compounds useful as transfer agents for use in compositions and methods disclosed herein are used, e. g. , as freshly made concentrations in the range of about 0.015 to about 2 percent by weight (wt percent) (e. g. , about 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045, 0.05, 0.055, 0.06, 0.065, 0.07, 0.075, 0.08, 0.085, 0.09, 0.095, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.5 wt percent).
[0059] Embodiments of transfer agents include one or more salts such as ammonium chloride, tetrabutylphosphonium bromide, and ammonium sulfate, provided in or used with a composition comprising an insecticidal polynucleotide disclosed herein. In some embodiments, ammonium chloride, tetrabutylphosphonium bromide, and/or ammonium sulfate are used at a concentration of about 0.5% to about 5% (w/v), or about 1% to about 3% (w/v), or about 2% (w/v). In certain embodiments, the composition comprising an insecticidal polynucleotide comprises an ammonium salt at a concentration greater or equal to 300 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide comprises an organosilicone transfer agent in a concentration of about 0.015 to about 2 percent by weight (wt percent) as well as ammonium sulfate at concentrations from about 80 to about 1200 millimolar or about 150 millimolar to about 600 millimolar.
[0060] Embodiments of transfer agents include a phosphate salt. Phosphate salts useful in a composition comprising an insecticidal polynucleotide include, but are not limited to, calcium, magnesium, potassium, or sodium phosphate salts. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a phosphate salt at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a phosphate salt in a range of about 1 millimolar to about 25 millimolar or in a range of about 5 millimolar to about 25 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes sodium phosphate at a concentration of at least about 5 millimolar, at least about 10 millimolar, or at least about 20 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes sodium phosphate at a concentration of about 5 millimolar, about 10 millimolar, or about 20 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a sodium phosphate salt in a range of about 1 millimolar to about 25 millimolar or in a range of about 5 millimolar to about 25 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a sodium phosphate salt in a range of about 10 millimolar to about 160 millimolar or in a range of about 20 millimolar to about 40 millimolar. In certain embodiments, the composition comprising an insecticidal polynucleotide includes a sodium phosphate buffer at a pH of about 6.8.
[0061] Embodiments of transfer agents include surfactants and/or effective molecules contained therein. Surfactants and/or effective molecules contained therein include, but are not limited to, sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organo silicone surfactants. In certain embodiments, the composition comprising an insecticidal polynucleotide is formulated with counter-ions or other molecules that are known to associate with nucleic acid molecules. Non-limiting examples include, tetraalkyl ammonium ions, trialkyl ammonium ions, sulfonium ions, lithium ions, and polyamines such as spermine, spermidine, or putrescine. In certain embodiments, the composition comprising an insecticidal polynucleotide is formulated with a non-polynucleotide herbicide e. g. , glyphosate, auxin-like benzoic acid herbicides including dicamba, chloramben, and TBA, glufosinate, auxin-like herbicides including phenoxy carboxylic acid herbicide, pyridine carboxylic acid herbicide, quinoline carboxylic acid herbicide, pyrimidine carboxylic acid herbicide, and benazolin-ethyl herbicide, sulfonylureas, imidazolinones, bromoxynil, delapon, cyclohezanedione, protoporphyrinogen oxidase inhibitors, and 4-hydroxyphenyl-pyruvate-dioxygenase inhibiting herbicides. In certain embodiments, the composition comprising an insecticidal
polynucleotide is formulated with a non-polynucleotide pesticide, e. g., a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein.
METHODS OF PROVIDING PLANTS WITH IMPROVED INSECT RESISTANCE
[0062] Several embodiments relate to a method of providing a plant having improved resistance to an insect, comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA construct comprises DNA encoding an insecticidal polynucleotidecomprising a sequence of at least 21 contiguous nucleotides that are essentially identical or essentially
complementary to a fragment of at least one insect target gene selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S 14, and 60S ribosomal protein L13. In some embodiments, the DNA construct comprises DNA encoding an insecticidal polynucleotide comprising a sequence essentially identical or essentially complementary to a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. Several embodiments relate to a plant produced by such method. In some embodiments, the DNA construct comprises DNA encoding an insecticidal polynucleotide having about 95% to about 100% (e. g. , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID
NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0063] In some embodiments, the recombinant DNA construct further comprises a heterologous promoter operably linked to the DNA encoding an insecticidal polynucleotide, wherein the heterologous promoter is functional in a plant cell. "Heterologous" refers to nucleic acid sequences that are not usually operably linked in a native or naturally occurring genome; by "heterologous promoter" is meant that the promoter is not natively operably linked with the DNA encoding an insecticidal polynucleotide. Promoters functional in a plant cell include those listed under the heading "Promoters".
[0064] In some embodiments, the recombinant DNA construct is expressed in the plant by means of transgenic expression or transient expression. In some embodiments, the method further comprises expression in the plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae,
Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae. The pesticidal agent can be expressed from the same recombinant DNA construct that comprises the DNA encoding an insecticidal polynucleotide, or from a different recombinant DNA construct.
[0065] A related aspect is a plant having improved resistance to an insect (e. g. , improved resistance to flea beetles), or the seed of such a plant, wherein the plant is provided by the method comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA construct comprises DNA encoding an insecticidal polynucleotide having a sequence essentially identical or essentially complementary to a fragment of at least one target gene of the insect, wherein the target gene is selected from the group consisting of actin, Act5C, arginine kinase, COPI (coatomer subunit) alpha, COPI (coatomer subunit) beta, COPI (coatomer subunit) betaPrime, COPI (coatomer subunit) delta, COPI (coatomer subunit) epsilon, COPI (coatomer subunit) gamma, COPI (coatomer subunit) zeta, RpL07, RpL19, RpL3, RpL40, RpL13, RpL14, RpS21, RpS4, RpS14, Rpn2, Rpn3, Rpn7, Rpt6, Rpn8, Rpn9, Rpn6-PB-like protein, Sari, sec6, sec23, sec23A, shrb (snf7), Tubulin gamma chain, ProsAlpha2, ProsBeta5, Proteasome alpha 2, Proteasome beta 5, VATPase E, VATPase A, VATPase B, VATPase D, Vps2, Vps4, Vpsl6A, Vps20, Vps24, Vps27, Vps28, Vha26 (V-ATPase A), Vha68-2 (V-ATPase D/E), 40S ribosomal protein S14, and 60S ribosomal protein L13. In some embodiments, the recombinant DNA construct comprises DNA encoding an insecticidal polynucleotide comprising a sequence essentially identical or essentially complementary to a fragment of a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859. In some embodiments, the plant exhibiting improved resistance to the insect is characterized by improved yield, when compared to a plant not expressing the recombinant DNA construct. In an embodiment, yield (oilseed biomass or oil content) in canola or oilseed rape plants is improved by expressing in the canola or oilseed rape plants an insecticidal polynucleotide, such as a dsRNA molecule, targetting one or more genes of Phyllotreta cruciferae (canola flea beetle), e. g. , wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532. Several embodiments relate to fruit, seed, or
propagatable parts of the plant provided by this method and exhibiting improved resistance to the insect. The plant can be any plant that is subject to infestation by an insect that can be controlled by expressing in the plant the recombinant DNA construct according to this method. Plants of particular interest include commercially important plants, including row crop plants, vegetables, and fruits, and other plants of agricultural or decorative use. Examples of suitable plants are provided under the heading "Plants". The method is especially useful for providing an ornamental plant or a crop plant with improved resistance to flea beetles. Various embodiments of the method include those wherein the plant is a plant in the family Brassicaceae, including a Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii, and B. fructiculosa. In other embodiments, the plant is selected from the group consisting of Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocus nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgar e, and Triticum aestivum. In an embodiment, the method provides a potato plant with improved resistance to Epitrix cucumeris (potato flea beetle).
[0066] Embodiments of the method provide a plant having improved resistance to one or more flea beetle species, e. g., a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis,
Lipromima, Liprus, Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia,
Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and Zipangia. In embodiments, the insect is selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle). In embodiments, the method provides a plant having improved resistance to an insect selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes punctulata (hop flea beetle).
[0067] In some embodiments, the method is developed for specific target genes in a given insect species. In some embodiments, a plant having improved resistance to Phyllotreta atra (turnip flea beetle), wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 296, is provided. In some embodiments, a plant having improved resistance to Phyllotreta cruciferae (canola flea beetle), wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532, is provided. In some embodiments, a plant having improved resistance to Phyllotreta striolata (striped flea beetle), wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551, is provided. In some embodiments, a plant having improved resistance to Psylliodes chrysocephala, wherein the target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859, is provided. In some embodiments, a plant having improved resistance to Phyllotreta atra (turnip flea beetle) that expresses from a recombinant DNA construct a dsRNA strand comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1155 or a fragment thereof is provided. In some embodiments, a plant having improved resistance to Phyllotreta cruciferae (canola flea beetle) that expresses from a recombinant DNA construct an insecticidal polynucleotide comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:1156 - 1391 , 1731-1972, and 1974 or a fragment thereof is provided. In some embodiments, a plant having improved resistance to Phyllotreta striolata (striped flea beetle) that expresses from a recombinant DNA construct an insecticidal polynucleotide comprising a strand comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975 or a fragment thereof is provided. In some embodiments, a plant having improved resistance to Psylliodes chrysocephala that expresses from a recombinant DNA construct an insecticidal polynucleotide comprising a strand comprising a sequence of about 95% to about 100% identity with a sequence selected from the group consisting of SEQ ID NOs:1411 -1718 or a fragment thereof is provided.
RECOMBINANT DNA CONSTRUCTS ENCODING INSECTICIDAL POLYNUCLEOTIDES FOR INSECT CONTROL
[0068] Several embodiments relate to a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an insecticidal polynucleotide transcript comprising a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity to at least 21 contiguos nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0069] In some embodiments of the recombinant DNA construct, the insecticidal polynucleotide transcript comprises a sequence of about 95% to about 100% (e. g , about 95%, about 96%, about 97%, about 98%, about 99%, or about 100%) identity or complementarity with at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript forms dsRNA double-stranded insecticidal polynucleotide. In some embodiments, the insecticidal polynucleotide transcript is a dsRNA comprising an RNA strand comprising at least one segment of 18 or more contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof In some embodiments, the insecticidal polynucleotide transcript is a dsRNA comprising an RNA strand comprising at least one segment of 21 contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a dsRNA comprising at least one RNA strand comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a dsRNA comprising an RNA strand comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript forms single-stranded insecticidal polynucleotide. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising at least one segment of 18 or more contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising at least one segment of 21 contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID
NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In some embodiments, the insecticidal polynucleotide transcript is a ssRNA comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof.
[0070] In some embodiments of the recombinant DNA construct, the heterologous promoter is functional for expression of the insecticidal polynucleotide transcript in a bacterium. In some embodiments where the recombinant DNA construct is to be expressed in a bacterium, the bacterium is selected from the group consisting of Escherichia coli, Bacillus species, Pseudomonas species,
Xenorhabdus species, or Photorhabdus species. In other embodiments, the recombinant DNA construct comprises a heterologous promoter that is functional in a plant cell. [0071] In some embodiments, the recombinant DNA construct is contained in a recombinant vector, such as a recombinant plant virus vector or a recombinant baculovirus vector. In embodiments, the recombinant DNA construct is integrated into a plant chromosome or plastid, e. g. , by stable transformation.
[0072] Related aspects include a transgenic plant cell comprising in its genome the recombinant DNA construct, and a transgenic plant comprising such a transgenic plant cell. Transgenic plant cells and plants are made by methods known in the art, such as those described under the heading "Making and Using Transgenic Plant Cells and Transgenic Plants". Further aspects include a commodity product produced from such a transgenic plant, and transgenic progeny seed or
propagatable plant part of the transgenic plant.
RELATED INFORMATION AND TECHNIQUES
Plants
[0073] The methods and compositions described herein for treating and protecting plants from insect infestations are useful across a broad range of plants. Suitable plants in which the methods and compositions disclosed herein can be used include, but are not limited to, cereals and forage grasses (rice, maize, wheat, barley, oat, sorghum, pearl millet, finger millet, cool-season forage grasses, and bahiagrass), oilseed crops (soybean, oilseed brassicas including canola and oilseed rape, sunflower, peanut, flax, sesame, and safflower), legume grains and forages (common bean, cowpea, pea, fava bean, lentil, tepary bean, Asiatic beans, pigeonpea, vetch, chickpea, lupine, alfalfa, and clovers), temperate fruits and nuts (apple, pear, peach, plums, berry crops, cherries, grapes, olive, almond, and Persian walnut), tropical and subtropical fruits and nuts (citrus including limes, oranges, and grapefruit; banana and plantain, pineapple, papaya, mango, avocado, kiwifruit, passionfruit, and persimmon), vegetable crops (solanaceous plants including tomato, eggplant, and peppers; vegetable brassicas; radish, carrot, cucurbits, alliums, asparagus, and leafy vegetables), sugar, tuber, and fiber crops
(sugarcane, sugar beet, stevia, potato, sweet potato, cassava, and cotton), plantation crops, ornamentals, and turf grasses (tobacco, coffee, cocoa, tea, rubber tree, medicinal plants, ornamentals, and turf grasses), and forest tree species. Specific plant species of interest are plants in the family Brassicaceae, including the Brassica species B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii, and B. fructiculosa. Additional plant species of interest are Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum. Additional Construct Elements
[0074] Embodiments of the polynucleotides and nucleic acid molecules disclosed herein can comprise additional elements, such as promoters, small RNA recognition sites, aptamers or ribozymes, additional and additional expression cassettes for expressing coding sequences (e. g. , to express a transgene such as an insecticidal protein or selectable marker) or non -coding sequences (e. g. , to express additional suppression elements). For example, an aspect provides a recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof. In another embodiment, a recombinant DNA construct comprising a promoter operably linked to DNA encoding: (a) an RNA transcript comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 or a fragment thereof, and (b) an aptamer, is stably integrated into the plant's genome from where RNA transcripts comprising the RNA aptamer and the RNA silencing element are expressed in cells of the plant; the aptamer serves to guide the RNA silencing element to a desired location in the cell. In another embodiment, inclusion of one or more recognition sites for binding and cleavage by a small RNA (e. g. , by a miRNA or an siRNA that is expressed only in a particular cell or tissue) allows for more precise expression patterns in a plant, wherein the expression of the
recombinant DNA construct is suppressed where the small RNA is expressed. Such additional elements are described below.
Promoters
[0075] Promoters of use in the compositions and methods disclosed herein are functional in the cell in which the construct is intended to be transcribed. Generally these promoters are
heterologous promoters, as used in recombinant constructs, i. e. , they are not in nature found to be operably linked to the other nucleic elements used in the constructs. In various embodiments, the promoter is selected from the group consisting of a constitutive promoter, a spatially specific promoter, a temporally specific promoter, a developmentally specific promoter, and an inducible promoter. In many embodiments the promoter is a promoter functional in a plant, for example, a pol II promoter, a pol III promoter, a pol IV promoter, or a pol V promoter.
[0076] Non-constitutive promoters suitable for use with the recombinant DNA constructs disclosed herein include spatially specific promoters, temporally specific promoters, and inducible promoters. Spatially specific promoters can include organelle-, cell-, tissue-, or organ-specific promoters (e. g. , a plastid-specific, a root-specific, a pollen-specific, or a seed-specific promoter for expression in plastids, roots, pollen, or seeds, respectively). In many cases a seed-specific, embryo- specific, aleurone-specific, or endosperm-specific promoter is especially useful. Temporally specific promoters can include promoters that tend to promote expression during certain developmental stages in a plant's growth cycle, or during different times of day or night, or at different seasons in a year. Inducible promoters include promoters induced by chemicals or by environmental conditions such as, but not limited to, biotic or abiotic stress (e. g. , water deficit or drought, heat, cold, high or low nutrient or salt levels, high or low light levels, or pest or pathogen infection). MicroR A promoters are useful, especially those having a temporally specific, spatially specific, or inducible expression pattern;
examples of miRNA promoters, as well as methods for identifying miRNA promoters having specific expression patterns, are provided in U. S. Patent Application Publications 2006/0200878,
2007/0199095, and 2007/0300329, which are specifically incorporated herein by reference. An expression-specific promoter can also include promoters that are generally constitutively expressed but at differing degrees or "strengths" of expression, including promoters commonly regarded as "strong promoters" or as "weak promoters".
[0077] Promoters include the following examples: an opaline synthase promoter isolated from T-DNA of Agrobacterium; a cauliflower mosaic virus 35S promoter; enhanced promoter elements or chimeric promoter elements such as an enhanced cauliflower mosaic virus (CaMV) 35 S promoter linked to an enhancer element (an intron from heat shock protein 70 of Zea mays); root specific promoters such as those disclosed in U.S. Patents 5,837,848; 6,437,217 and 6,426,446; a maize L3 oleosin promoter disclosed in U.S. Patent 6,433,252; a promoter for a plant nuclear gene encoding a plastid-localized aldolase disclosed in U. S. Patent Application Publication 2004/0216189; cold- inducible promoters disclosed in U.S. Patent 6,084,089; salt-inducible promoters disclosed in U. S. Patent Number 6, 140,078; light-inducible promoters disclosed in U.S. Patent 6,294,714; pathogen- inducible promoters disclosed in U.S. Patent 6,252, 138; and water deficit-inducible promoters disclosed in U.S. Patent Application Publication 2004/0123347 Al . All of the above-described patents and patent publications disclosing promoters and their use, especially in recombinant DNA constructs functional in plants are incorporated herein by reference.
[0078] Plant vascular- or phloem-specific promoters of interest include a rolC or rolA promoter of Agrobacterium rhizogenes, a promoter of a. Agrobacterium tumefaciens T-DNA gene 5, the rice sucrose synthase RSsl gene promoter, a Commelina yellow mottle badnavirus promoter, a coconut foliar decay virus promoter, a rice tungro bacilliform virus promoter, the promoter of a pea glutamine synthase GS3A gene, a invCD l 1 1 and invCD 141 promoters of a potato invertase genes, a promoter isolated from Arabidopsis shown to have phloem-specific expression in tobacco by Kertbundit et al. (1991) Proc. Natl. Acad. Sci. USA., 88:5212-5216, a VAHOX1 promoter region, a pea cell wall invertase gene promoter, an acid invertase gene promoter from carrot, a promoter of a sulfate transporter gene Sultrl;3, a promoter of a plant sucrose synthase gene, and a promoter of a plant sucrose transporter gene.
[0079] Promoters suitable for use with a recombinant DNA construct or polynucleotide disclosed herein include polymerase II ("pol II") promoters and polymerase III ("pol III") promoters. RNA polymerase II transcribes structural or catalytic RNAs that are usually shorter than 400 nucleotides in length, and recognizes a simple run of T residues as a termination signal; it has been used to transcribe siRNA duplexes (see, e. g. , Lu et al. (2004) Nucleic Acids Res., 32:e l71). Pol II promoters are therefore preferred in certain embodiments where a short RNA transcript is to be produced from a recombinant DNA construct. In one embodiment, the recombinant DNA construct includes a pol II promoter to express an R A transcript flanked by self-cleaving ribozyme sequences (e. g , self-cleaving hammerhead ribozymes), resulting in a processed RNA, such as a single-stranded RNA that binds to the transcript of the flea beetle target gene, with defined 5 ' and 3 ' ends, free of potentially interfering flanking sequences. An alternative approach uses pol III promoters to generate transcripts with relatively defined 5' and 3' ends, /'. e. , to transcribe an RNA with minimal 5' and 3' flanking sequences. In some embodiments, Pol III promoters (e. g. , U6 or HI promoters) are preferred for adding a short AT-rich transcription termination site that results in 2 base-pair overhangs (UU) in the transcribed RNA; this is useful, e. g , for expression of siRNA-type constructs. Use of pol III promoters for driving expression of siRNA constructs has been reported; see van de Wetering et al. (2003) EMBO Rep., 4: 609-615, and Tuschl (2002) Nature Biotechnol, 20: 446-448. Baculovirus promoters such as baculovirus polyhedrin and p 10 promoters are known in the art and commercially available; see, e. g , Invitrogen's "Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques", 2002 (Life Technologies, Carlsbad, CA) and F. J. Haines et al. "Baculovirus Expression Vectors", undated (Oxford Expression Technologies, Oxford, UK).
[0080] The promoter element can include nucleic acid sequences that are not naturally occurring promoters or promoter elements or homologues thereof but that can regulate expression of a gene. Examples of such "gene independent" regulatory sequences include naturally occurring or artificially designed RNA sequences that comprise a ligand-binding region or aptamer (see "Aptamers", below) and a regulatory region (which can be cz's -acting). See, for example, Isaacs et al. (2004) Nat. Biotechnol, 22:841-847, Bayer and Smolke (2005) Nature Biotechnol, 23:337-343, Mandal and Breaker (2004) Nature Rev. Mol. Cell Biol, 5:451-463, Davidson and Ellington (2005) Trends
Biotechnol, 23: 109-112, Winkler et al. (2002) Nature, 419:952-956, Sudarsan et al. (2003) RNA, 9:644-647, and Mandal and Breaker (2004) Nature Struct. Mol. Biol, 11 :29-35. Such "riboregulators" could be selected or designed for specific spatial or temporal specificity, for example, to regulate translation of DNA that encodes a silencing element for suppressing a target gene only in the presence (or absence) of a given concentration of the appropriate ligand. One example is a riboregulator that is responsive to an endogenous ligand (e. g , jasmonic acid or salicylic acid) produced by the plant when under stress (e. g. , abiotic stress such as water, temperature, or nutrient stress, or biotic stress such as attach by pests or pathogens); under stress, the level of endogenous ligand increases to a level sufficient for the riboregulator to begin transcription of the DNA that encodes a silencing element for suppressing a target gene.
Transgene Transcription Units
[0081] In some embodiments, the recombinant DNA construct or polynucleotide disclosed herein comprisies a transgene transcription unit. A transgene transcription unit comprises DNA sequence encoding a gene of interest, e. g. , a natural protein or a heterologous protein. A gene of interest can be any coding or non-coding sequence from any species (including, but not limited to, non- eukaryotes such as bacteria, and viruses; fungi, protists, plants, invertebrates, and vertebrates. Particular genes of interest are genes encoding one or more proteins conferring resistance to an herbicide and genes encoding at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a. Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and an insecticidal protein produced by any of Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus (Bacillus later osporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and
Paenibacillus popilliae. The transgene transcription unit can further comprise 5' or 3' sequence or both as required for transcription of the transgene.
Introns
[0082] In some embodiments, the recombinant DNA construct or polynucleotide comprises DNA encoding a spliceable intron. By "intron" is generally meant a segment of DNA (or the RNA transcribed from such a segment) that is located between exons (protein-encoding segments of the DNA or corresponding transcribed RNA), wherein, during maturation of the messenger RNA, the intron present is enzymatically "spliced out" or removed from the RNA strand by a cleavage/ligation process that occurs in the nucleus in eukaryotes. The term "intron" is also applied to non-coding DNA sequences that are transcribed to RNA segments that can be spliced out of a maturing RNA transcript, but are not introns found between protein-coding exons. One example of these are spliceable sequences that that have the ability to enhance expression in plants (in some cases, especially in monocots) of a downstream coding sequence; these spliceable sequences are naturally located in the 5' untranslated region of some plant genes, as well as in some viral genes (e. g. , the tobacco mosaic virus 5' leader sequence or "omega" leader described as enhancing expression in plant genes by Gallie and Walbot (1992) Nucleic Acids Res., 20:4631-4638). These spliceable sequences or "expression-enhancing introns" can be artificially inserted in the 5' untranslated region of a plant gene between the promoter but before any protein-coding exons. Examples of such expression-enhancing introns include, but are not limited to, a maize alcohol dehydrogenase (Z -Adhl), a maize Bronze-1 expression-enhancing intron, a rice actin 1 (Os-Actl) intron, a Shrunken- 1 (Sh-1) intron, a maize sucrose synthase intron, a heat shock protein 18 (hsplS) intron, and an 82 kilodalton heat shock protein (hsp82) intron. U. S. Patents 5,593,874 and 5,859,347, specifically incorporated by reference herein, describe methods of improving recombinant DNA constructs for use in plants by inclusion of an expression-enhancing intron derived from the 70 kilodalton maize heat shock protein (hsp70) in the non-translated leader positioned 3' from the gene promoter and 5' from the first protein-coding exon.
Gene Suppression Elements
[0083] In some embodiments, the recombinant DNA construct or polynucleotide comprises DNA encoding additional gene suppression element for suppressing a target gene other than a flea beetle target gene. The target gene to be suppressed can include coding or non-coding sequence or both. [0084] Suitable gene suppression elements are described in detail in U. S. Patent Application Publication 2006/0200878, which disclosure is specifically incorporated herein by reference, and include one or more of:
(a) DNA that comprises at least one anti -sense DNA segment that is anti -sense to at least one segment of the gene to be suppressed;
(b) DNA that comprises multiple copies of at least one anti-sense DNA segment that is anti- sense to at least one segment of the gene to be suppressed;
(c) DNA that comprises at least one sense DNA segment that is at least one segment of the gene to be suppressed;
(d) DNA that comprises multiple copies of at least one sense DNA segment that is at least one segment of the gene to be suppressed;
(e) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming double- stranded RNA and comprises at least one anti-sense DNA segment that is anti-sense to at least one segment of the gene to be suppressed and at least one sense DNA segment that is at least one segment of the gene to be suppressed;
(f) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming a single double -stranded RNA and comprises multiple serial anti-sense DNA segments that are anti- sense to at least one segment of the gene to be suppressed and multiple serial sense DNA segments that are at least one segment of the gene to be suppressed;
(g) DNA that transcribes to RNA for suppressing the gene to be suppressed by forming
multiple double strands of RNA and comprises multiple anti -sense DNA segments that are anti-sense to at least one segment of the gene to be suppressed and multiple sense DNA segments that are at least one segment of the gene to be suppressed, and wherein the multiple anti-sense DNA segments and the multiple sense DNA segments are arranged in a series of inverted repeats;
(h) DNA that comprises nucleotides derived from a plant miRNA;
(i) DNA that comprises nucleotides of a siRNA;
(j) DNA that transcribes to an RNA aptamer capable of binding to a ligand; and
(k) DNA that transcribes to an RNA aptamer capable of binding to a ligand, and DNA that transcribes to regulatory RNA capable of regulating expression of the gene to be suppressed, wherein the regulation is dependent on the conformation of the regulatory RNA, and the conformation of the regulatory RNA is allosterically affected by the binding state of the RNA aptamer.
[0085] In some embodiments, an intron is used to deliver a gene suppression element in the absence of any protein-coding exons (coding sequence). In one example, an intron, such as an expression-enhancing intron (preferred in certain embodiments), is interrupted by embedding within the intron a gene suppression element, wherein, upon transcription, the gene suppression element is excised from the intron. Thus, protein-coding exons are not required to provide the gene suppressing function of the recombinant DNA constructs disclosed herein.
Transcription Regulatory Elements
[0086] In some embodiments, the recombinant DNA construct or polynucleotide comprises DNA encoding a transcription regulatory element. Transcription regulatory elements include elements that regulate the expression level of the recombinant DNA construct (relative to its expression in the absence of such regulatory elements). Examples of suitable transcription regulatory elements include riboswitches (cis- or fra«s-acting), transcript stabilizing sequences, and miRNA recognition sites, as described in detail in U. S. Patent Application Publication 2006/0200878, specifically incorporated herein by reference .
Transgenic Plant Cells and Transgenic Plants
[0087] The recombinant DNA constructs disclosed herein can be stacked with other recombinant DNA for imparting additional traits (e. g. , in the case of transformed plants, traits including herbicide resistance, pest resistance, cold germination tolerance, water deficit tolerance, and the like) for example, by expressing or suppressing other genes. Constructs for coordinated decrease and increase of gene expression are disclosed in U.S. Patent Application Publication 2004/0126845 Al, specifically incorporated by reference.
[0088] In certain transgenic plant cells and transgenic plants, it is sometimes desirable to concurrently express a gene of interest while also modulating expression of a flea beetle target gene. Thus, in some embodiments, the transgenic plant contains recombinant DNA further comprising a gene expression element for expressing at least one gene of interest, and transcription of the recombinant DNA construct for flea beetle control is preferably effected with concurrent transcription of the gene expression element. In embodiments, the transgenic plant expresses DNA encoding a insecticidal polynucleotide transcript as disclosed herein for suppression of a flea beetle target gene, and also expresses DNA encoding a non-nucleotide pesticidal agent such as a small-molecule pesticidal agent or a proteinaceous pesticidal agent; such DNAs can be stacked in a single recombinant construct or expression cassette, or alternatively can be expressed from discrete recombinant constructs or expression cassettes. Examples of non-nucleotide pesticidal agents include patatins, plant lectins, phytoecdysteroids, and bacterial insecticidal proteins (e. g. , insecticidal proteins from Bacillus thuringiensis, Xenorhabdus sp., Photorhabdus sp., Brevibacillus laterosporus {Bacillus later osporus), Lysinibacillus sphaericus {Bacillus sphaericus), Chromobacterium sp., Chromobacterium subtsugae, Paenibacillus sp., Paenibacillus lentimorbus, and Paenibacillus popilliae). In embodiments, the transgenic plant expresses DNA encoding a recombinant RNA transcript as disclosed herein for suppression of a flea beetle target gene, and also expresses DNA encoding one or more proteins conferring tolerance to an herbicide. Examples of proteins conferring tolerance to an herbicide include 5 -enolpyruvylshikimate-3 -phosphate synthases (EPSPS; see, e. g. , U. S. Patent Numbers 5,627,061, 5,633,435 RE39247, 6,040,497, and 5,094,945, and PCT International Application Publications WO04074443 and WO04009761), glyphosate oxidoreductase (GOX; U. S. Patent Number 5,463, 175), glyphosate decarboxylase (PCT International Application Publication WO05003362, U. S. Patent Number 7,405,347, and U. S. Patent Application Publication 2004/0177399), glyphosate-N-acetyl transferase (GAT; U. S. Patent Number 7,714, 188) conferring tolerance to glyphosate; dicamba monooxygenase conferring tolerance to auxin -like herbicides such as dicamba (U. S. Patent Number 7, 105,724); phosphinothricin acetyltransferase (pat or bar) conferring tolerance to phosphinothricin or glufosinate (U. S. Patent Number 5,646,024); 2,2- dichloropropionic acid dehalogenase conferring tolerance to 2,2-dichloropropionic acid (Dalapon) (PCT International Application Publication
W099271 16); acetohydroxyacid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide (U. S. Patent Number 6,225, 105); haloarylnitrilase (Bxn) for conferring tolerance to bromoxynil (U. S. Patent Number 4,810,648); modified acetyl-coenzyme A carboxylase for conferring tolerance to cyclohexanedione (sethoxydim) and aryloxyphenoxypropionate (haloxyfop) (U. S. Patent Number 6,414,222); dihydropteroate synthase (sul I) for conferring tolerance to sulfonamide herbicides (U. S. Patent Number 5,719,046); 32 kDa photosystem II polypeptide (psbA) for conferring tolerance to triazine herbicides (Hirschberg et al , 1983, Science, 222: 1346-1349); anthranilate synthase for conferring tolerance to 5-methyltryptophan (U. S. Patent Number 4,581,847); dihydrodipicolinic acid synthase (dap A) for conferring to tolerance to aminoethyl cysteine (PCT International Application Publication W0891 1789); phytoene desaturase (crtl) for conferring tolerance to pyridazinone herbicides such as norflurazon (Japan Patent JP06343473); hydroxyphenylpyruvate dioxygenase, a 4- hydroxyphenylacetic acid oxidase and a 4-hydroxyphenylacetic 1 -hydrolase (US Patent 7,304,209) for conferring tolerance to cyclopropylisoxazole herbicides such as isoxaflutole (U. S. Patent Number 6,268,549); modified protoporphyrinogen oxidase I (protox) for conferring tolerance to
protoporphyrinogen oxidase inhibitors (U. S. Patent Number 5939602); aryloxyalkanoate dioxygenase (AAD-1) for conferring tolerance to an herbicide containing an aryloxyalkanoate moiety
(WO05107437); a serine hydroxymethyltransferase (US Patent Application Publication 2008/0155716), a glufosinate-tolerant glutamine synthase (US Patent Application Publication 2009/0018016).
Examples of such herbicides include phenoxy auxins (such as 2,4- D and dichlorprop), pyridyloxy auxins (such as fluroxypyr and triclopyr), aryloxyphenoxypropionates (AOPP) acetylcoenzyme A carboxylase (ACCase) inhibitors (such as haloxyfop, quizalofop, and diclofop), and 5- substituted phenoxyacetate protoporphyrinogen oxidase IX inhibitors (such as pyraflufen and flumiclorac). The nucleotide sequences of the nucleic acids encoding herbicide-tolerance proteins and the sequences of the herbicide-tolerance proteins, as disclosed in the U. S. patent and patent application publications cited in this paragraph are incorporated herein by reference.
[0089] In some embodiments, the recombinant DNA constructs disclosed herein can be transcribed in any plant cell or tissue or in a whole plant of any developmental stage. Transgenic plants can be derived from any monocot or dicot plant, such as, but not limited to, plants of commercial or agricultural interest, such as crop plants (especially crop plants used for human food or animal feed), wood- or pulp-producing trees, vegetable plants, fruit plants, and ornamental plants. Examples of plants of interest include grain crop plants (such as wheat, oat, barley, maize, rye, triticale, rice, millet, sorghum, quinoa, amaranth, and buckwheat); forage crop plants (such as forage grasses and forage dicots including alfalfa, vetch, clover, and the like); oilseed crop plants (such as cotton, safflower, sunflower, soybean, canola, rapeseed, flax, peanuts, and oil palm); tree nuts (such as walnut, cashew, hazelnut, pecan, almond, and the like); sugarcane, coconut, date palm, olive, sugarbeet, tea, and coffee; wood- or pulp-producing trees; vegetable crop plants such as legumes (for example, beans, peas, lentils, alfalfa, peanut), lettuce, asparagus, artichoke, celery, carrot, radish, the brassicas (for example, cabbages, kales, mustards, and other leafy brassicas, broccoli, cauliflower, Brussels sprouts, turnip, kohlrabi), edible cucurbits (for example, cucumbers, melons, summer squashes, winter squashes), edible alliums (for example, onions, garlic, leeks, shallots, chives), edible members of the Solanaceae (for example, tomatoes, eggplants, potatoes, peppers, groundcherries), and edible members of the Chenopodiaceae (for example, beet, chard, spinach, quinoa, amaranth); fruit crop plants such as apple, pear, citrus fruits (for example, orange, lime, lemon, grapefruit, and others), stone fruits (for example, apricot, peach, plum, nectarine), banana, pineapple, grape, kiwifruit, papaya, avocado, and berries; plants grown for biomass or biofuel (for example, Miscanthus grasses, switchgrass, jatropha, oil palm, eukaryotic microalgae such as Botryococcus braunii, Chlorella spp., and Dunaliella spp., and eukaryotic macroalgae such as Gracilaria spp., and Sargassum spp.); and ornamental plants including ornamental flowering plants, ornamental trees and shrubs, ornamental groundcovers, and ornamental grasses. Specific plant species of interest in which a recombinant DNA construct is transcribed to provide resistance to flea beetles are plants in the family Brassicaceae, including the Brassica species B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii, and B. fructiculosa. Additional plant species of interest in which a recombinant DNA construct is transcribed to provide resistance to flea beetles are Glycine max, Linum usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum.
[0090] Also disclosed herein are commodity products produced from a transgenic plant cell, plant, or seed expressing a recombinant DNA construct imparting improved resistance to flea beetles as disclosed herein, including, but not limited to, harvested leaves, roots, shoots, tubers, stems, fruits, seeds, or other parts of a plant, meals, oils, extracts, fermentation or digestion products, crushed or whole grains or seeds of a plant, or any food or non-food product including such commodity products produced from a transgenic plant cell, plant, or seed as disclosed herein. The detection of one or more of nucleic acid sequences of the recombinant DNA constructs for flea beetle control as disclosed herein in one or more commodity or commodity products contemplated herein is de facto evidence that the commodity or commodity product contains or is derived from a transgenic plant cell, plant, or seed expressing such a recombinant DNA construct. [0091] Generally a transgenic plant having in its genome a recombinant DNA construct as disclosed herein exhibits increased resistance to an insect infestation, specifically increased resistance to a flea beetle infestation. In various embodiments, for example, where the transgenic plant expresses a recombinant DNA construct for flea beetle control that is stacked with other recombinant DNA for imparting additional traits, the transgenic plant has at least one additional altered trait, relative to a plant lacking the recombinant DNA construct, selected from the group of traits consisting of:
(a) improved abiotic stress tolerance;
(b) improved biotic stress tolerance;
(c) modified primary metabolite composition;
(d) modified secondary metabolite composition;
(e) modified trace element, carotenoid, or vitamin composition;
(f) improved yield;
(g) improved ability to use nitrogen, phosphate, or other nutrients;
(h) modified agronomic characteristics;
(i) modified growth or reproductive characteristics; and
(j) improved harvest, storage, or processing quality.
[0092] In some embodiments, the transgenic plant is characterized by: improved tolerance of abiotic stress (e. g. , tolerance of water deficit or drought, heat, cold, non-optimal nutrient or salt levels, non-optimal light levels) or of biotic stress (e. g. , crowding, allelopathy, or wounding); by a modified primary metabolite (e. g. , fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition; a modified secondary metabolite (e. g. , alkaloids, terpenoids, polyketides, non-ribosomal peptides, and secondary metabolites of mixed biosynthetic origin) composition; a modified trace element (e. g. , iron, zinc), carotenoid (e. g. , beta-carotene, lycopene, lutein, zeaxanthin, or other carotenoids and xanthophylls), or vitamin (e. g. , tocopherols) composition; improved yield (e. g. , improved yield under non-stress conditions or improved yield under biotic or abiotic stress); improved ability to use nitrogen, phosphate, or other nutrients; modified agronomic characteristics (e. g. , delayed ripening; delayed senescence; earlier or later maturity; improved shade tolerance; improved resistance to root or stalk lodging; improved resistance to "green snap" of stems; modified photoperiod response); modified growth or reproductive characteristics (e. g. , intentional dwarfing; intentional male sterility, useful, e. g. , in improved hybridization procedures; improved vegetative growth rate; improved germination; improved male or female fertility); improved harvest, storage, or processing quality (e. g. , improved resistance to pests during storage, improved resistance to breakage, improved appeal to consumers); or any combination of these traits.
[0093] In another embodiment, transgenic seed, or seed produced by the transgenic plant, has modified primary metabolite (e. g. , fatty acid, oil, amino acid, protein, sugar, or carbohydrate) composition, a modified secondary metabolite composition, a modified trace element, carotenoid, or vitamin composition, an improved harvest, storage, or processing quality, or a combination of these. In another embodiment, it can be desirable to change levels of native components of the transgenic plant or seed of a transgenic plant, for example, to decrease levels of an allergenic protein or glycoprotein or of a toxic metabolite.
EXAMPLES
Example 1
[0094] This example illustrates non-limiting embodiments of coding DNA sequences useful as target genes for controlling insect species and for making compositions for controlling insects and insect-resistant transgenic plants, and identifies insecticidal polynucleotide sequences useful for controlling insect species. More specifically, embodiments of target genes identified by name (annotation) and sequence identifier (SEQ ID NO.) for controlling flea beetles are provided in SEQ ID NOs:l - 859, and embodiments of dsRNA sequences ranging in size from 135 to 352 base pairs and designed to suppress these target genes are provided in SEQ ID NOs.:860 - 1718.
Table 1
Figure imgf000045_0001
892 248 1107 463 1322 678 1537
893 249 1108 464 1323 679 1538
894 250 1109 465 1324 680 1539
895 251 1110 466 1325 681 1540
896 252 1111 467 1326 682 1541
897 253 1112 468 1327 683 1542
898 254 1113 469 1328 684 1543
899 255 1114 470 1329 685 1544
900 256 1115 471 1330 686 1545
901 257 1116 472 1331 687 1546
902 258 1117 473 1332 688 1547
903 259 1118 474 1333 689 1548
904 260 1119 475 1334 690 1549
905 261 1120 476 1335 691 1550
906 262 1121 477 1336 692 1551
907 263 1122 478 1337 693 1552
908 264 1123 479 1338 694 1553
909 265 1124 480 1339 695 1554
910 266 1125 481 1340 696 1555
911 267 1126 482 1341 697 1556
912 268 1127 483 1342 698 1557
913 269 1128 484 1343 699 1558
914 270 1129 485 1344 700 1559
915 271 1130 486 1345 701 1560
916 272 1131 487 1346 702 1561
917 273 1132 488 1347 703 1562
918 274 1133 489 1348 704 1563
919 275 1134 490 1349 705 1564
920 276 1135 491 1350 706 1565
921 277 1136 492 1351 707 1566
922 278 1137 493 1352 708 1567
923 279 1138 494 1353 709 1568
924 280 1139 495 1354 710 1569
925 281 1140 496 1355 711 1570
926 282 1141 497 1356 712 1571
927 283 1142 498 1357 713 1572
928 284 1143 499 1358 714 1573
929 285 1144 500 1359 715 1574
930 286 1145 501 1360 716 1575
931 287 1146 502 1361 717 1576
932 288 1147 503 1362 718 1577
933 289 1148 504 1363 719 1578
934 290 1149 505 1364 720 1579
935 291 1150 506 1365 721 1580
936 292 1151 507 1366 722 1581
937 293 1152 508 1367 723 1582
938 294 1153 509 1368 724 1583
939 295 1154 510 1369 725 1584
940 296 1155 511 1370 726 1585
941 297 1156 512 1371 727 1586
942 298 1157 513 1372 728 1587
943 299 1158 514 1373 729 1588
944 300 1159 515 1374 730 1589
945 301 1160 516 1375 731 1590 87 946 302 1161 517 1376 732 1591
88 947 303 1162 518 1377 733 1592
89 948 304 1163 519 1378 734 1593
90 949 305 1164 520 1379 735 1594
91 950 306 1165 521 1380 736 1595
92 951 307 1166 522 1381 737 1596
93 952 308 1167 523 1382 738 1597
94 953 309 1168 524 1383 739 1598
95 954 310 1169 525 1384 740 1599
96 955 311 1170 526 1385 741 1600
97 956 312 1171 527 1386 742 1601
98 957 313 1172 528 1387 743 1602
99 958 314 1173 529 1388 744 1603
100 959 315 1174 530 1389 745 1604
101 960 316 1175 531 1390 746 1605
102 961 317 1176 532 1391 747 1606
103 962 318 1177 533 1392 748 1607
104 963 319 1178 534 1393 749 1608
105 964 320 1179 535 1394 750 1609
106 965 321 1180 536 1395 751 1610
107 966 322 1181 537 1396 752 1611
108 967 323 1182 538 1397 753 1612
109 968 324 1183 539 1398 754 1613
110 969 325 1184 540 1399 755 1614
111 970 326 1185 541 1400 756 1615
112 971 327 1186 542 1401 757 1616
113 972 328 1187 543 1402 758 1617
114 973 329 1188 544 1403 759 1618
115 974 330 1189 545 1404 760 1619
116 975 331 1190 546 1405 761 1620
117 976 332 1191 547 1406 762 1621
118 977 333 1192 548 1407 763 1622
119 978 334 1193 549 1408 764 1623
120 979 335 1194 550 1409 765 1624
121 980 336 1195 551 1410 766 1625
122 981 337 1196 552 1411 767 1626
123 982 338 1197 553 1412 768 1627
124 983 339 1198 554 1413 769 1628
125 984 340 1199 555 1414 770 1629
126 985 341 1200 556 1415 771 1630
127 986 342 1201 557 1416 772 1631
128 987 343 1202 558 1417 773 1632
129 988 344 1203 559 1418 774 1633
130 989 345 1204 560 1419 775 1634
131 990 346 1205 561 1420 776 1635
132 991 347 1206 562 1421 777 1636
133 992 348 1207 563 1422 778 1637
134 993 349 1208 564 1423 779 1638
135 994 350 1209 565 1424 780 1639
136 995 351 1210 566 1425 781 1640
137 996 352 1211 567 1426 782 1641
138 997 353 1212 568 1427 783 1642
139 998 354 1213 569 1428 784 1643
140 999 355 1214 570 1429 785 1644 141 1000 356 1215 571 1430 786 1645
142 1001 357 1216 572 1431 787 1646
143 1002 358 1217 573 1432 788 1647
144 1003 359 1218 574 1433 789 1648
145 1004 360 1219 575 1434 790 1649
146 1005 361 1220 576 1435 791 1650
147 1006 362 1221 577 1436 792 1651
148 1007 363 1222 578 1437 793 1652
149 1008 364 1223 579 1438 794 1653
150 1009 365 1224 580 1439 795 1654
151 1010 366 1225 581 1440 796 1655
152 1011 367 1226 582 1441 797 1656
153 1012 368 1227 583 1442 798 1657
154 1013 369 1228 584 1443 799 1658
155 1014 370 1229 585 1444 800 1659
156 1015 371 1230 586 1445 801 1660
157 1016 372 1231 587 1446 802 1661
158 1017 373 1232 588 1447 803 1662
159 1018 374 1233 589 1448 804 1663
160 1019 375 1234 590 1449 805 1664
161 1020 376 1235 591 1450 806 1665
162 1021 377 1236 592 1451 807 1666
163 1022 378 1237 593 1452 808 1667
164 1023 379 1238 594 1453 809 1668
165 1024 380 1239 595 1454 810 1669
166 1025 381 1240 596 1455 811 1670
167 1026 382 1241 597 1456 812 1671
168 1027 383 1242 598 1457 813 1672
169 1028 384 1243 599 1458 814 1673
170 1029 385 1244 600 1459 815 1674
171 1030 386 1245 601 1460 816 1675
172 1031 387 1246 602 1461 817 1676
173 1032 388 1247 603 1462 818 1677
174 1033 389 1248 604 1463 819 1678
175 1034 390 1249 605 1464 820 1679
176 1035 391 1250 606 1465 821 1680
177 1036 392 1251 607 1466 822 1681
178 1037 393 1252 608 1467 823 1682
179 1038 394 1253 609 1468 824 1683
180 1039 395 1254 610 1469 825 1684
181 1040 396 1255 611 1470 826 1685
182 1041 397 1256 612 1471 827 1686
183 1042 398 1257 613 1472 828 1687
184 1043 399 1258 614 1473 829 1688
185 1044 400 1259 615 1474 830 1689
186 1045 401 1260 616 1475 831 1690
187 1046 402 1261 617 1476 832 1691
188 1047 403 1262 618 1477 833 1692
189 1048 404 1263 619 1478 834 1693
190 1049 405 1264 620 1479 835 1694
191 1050 406 1265 621 1480 836 1695
192 1051 407 1266 622 1481 837 1696
193 1052 408 1267 623 1482 838 1697
194 1053 409 1268 624 1483 839 1698 195 1054 410 1269 625 1484 840 1699
196 1055 411 1270 626 1485 841 1700
197 1056 412 1271 627 1486 842 1701
198 1057 413 1272 628 1487 843 1702
199 1058 414 1273 629 1488 844 1703
200 1059 415 1274 630 1489 845 1704
201 1060 416 1275 631 1490 846 1705
202 1061 417 1276 632 1491 847 1706
203 1062 418 1277 633 1492 848 1707
204 1063 419 1278 634 1493 849 1708
205 1064 420 1279 635 1494 850 1709
206 1065 421 1280 636 1495 851 1710
207 1066 422 1281 637 1496 852 1711
208 1067 423 1282 638 1497 853 1712
209 1068 424 1283 639 1498 854 1713
210 1069 425 1284 640 1499 855 1714
211 1070 426 1285 641 1500 856 1715
212 1071 427 1286 642 1501 857 1716
213 1072 428 1287 643 1502 858 1717
214 1073 429 1288 644 1503 859 1718
215 1074 430 1289 645 1504
*RNA sequences are provided for the anti-sense strand of the dsRNA in 5' to 3' direction.
** T44966 and T44967 are positive controls based on a Phyllotreta striolata arginine kinase mR A disclosed in Zhao et al. (2008), Eur. J. Entomol, 5:815.
[0095] The embodiments of dsRNA sequences provided in Table 1 are generally useful for
RNA -mediated suppression of the corresponding target gene identified in Table 1. These dsRNAs are useful for controlling insects, especially flea beetles, including the source species from which the target genes in Table 1 were identified. RNA-mediated suppression of one or more of the target genes provided in Table 1, or use of one or more of the dsRNAs provided in Table 1, is useful for causing mortality or stunting, or otherwise controlling, target insect species in the following genera: Altica,
Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda, Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis, Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus,
Longitarsus , Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila,
Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and Zipangia. In embodiments, compositions comprising a dsRNA for suppression of one or more of the target genes provided in Table 1 (e. g. , a composition comprising an effective amount of one or more of the dsRNAs provided in Table 1) are useful for controlling at least one of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle), thus preventing or treating plant infestation by these species. For example, a composition comprising an effective amount of one or more of the dsRNAs provided in Table 1 is useful for preventing or treating infestation of potato plants by Epitrix cucumeris (potato flea beetle).
[0096] In embodiments, RNA-mediated suppression of one or more of the target genes provided in Table 1, or use of one or more of the dsRNAs provided in Table 1, is useful for causing mortality or stunting in flea beetle species in the genera Phyllotreta and Psylliodes, thus preventing or treating plant infestation by these species. In specific embodiments, RNA-mediated suppression of one or more of the target genes provided in Table 1, or use of one or more of the dsRNAs provided in Table 1, is useful for causing mortality or stunting in at least one flea beetle species selected from the group consisting of Phyllotreta armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes punctulata (hop flea beetle). In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs: 1 - 296 is used to cause mortality or stunting in Phyllotreta atra (turnip flea beetle) adults or larvae, for example, by contacting Phyllotreta atra adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:860 - 1155. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs:297 - 532 is used to cause mortality or stunting in Phyllotreta cruciferae (canola flea beetle) adults or larvae, for example, by contacting Phyllotreta cruciferae adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731-1972, and 1974. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs:533 - 551 is used to cause mortality or stunting in Phyllotreta striolata (striped flea beetle) adults or larvae, for example, by contacting Phyllotreta striolata adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975. In embodiments, RNA-mediated suppression of one or more of the target genes having a sequence selected from the group consisting of SEQ ID NOs:552 - 859 is used to cause mortality or stunting in Psylliodes chrysocephala adults or larvae, for example, by contacting Psylliodes chrysocephala adults, larvae, or eggs with an effective amount of a dsRNA comprising a sequence selected from the group consisting of SEQ ID NOs:1411 - 1718.
[0097] Plants which can be protected by such infestation by transgenic expression or topical application of one or more of the dsRNAs provided in Table 1 include any plant species or variety that is subject to infestation by flea beetles, especially plants of economic importance, including ornamental plants and crop plants. Embodiments of such plants include plants in the family Brassicaceae (mustard family), such as a plant in the genus Brassica including, for example, one of the following: B. napus (rapeseed, including cultivars such as canola and rutabaga), B. juncea (Indian mustard), B. carinata (Abyssinian mustard), B. rapa (turnip), B. oleracea (wild cabbage, including domesticated cultivars such as, kale, cabbage, broccoli, cauliflower, brussels sprouts, etc.) B. rupestris (brown mustard), B. septiceps (seventop mustard), B. nigra (black mustard), B. narinosa (broadbeaked mustard), B.
perviridus (mustard spinach), B. tournefortii (asian mustard), and B. fructiculosa (Mediterranean cabbage). In additional embodiments, the target plants may include, but not limited to, one of the following: Glycine max (soybean), Linum usitatissimum (linseed/flax), Zea mays (maize), Carthamus tinctorius (safflower), Helianthus annuus (sunflower), Nicotiana tabacum (tobacco), Arabidopsis thaliana, Betholettia excelsa (Brazil nut), Ricinus communis (castor bean), Cocos nucifera (coconut), Coriandrum sativum (coriander), Gossypium spp. (cotton), Arachis hypogaea (groundnut or peanut), Simmondsia chinensis (jojoba), Solanum tuberosum (potato) Elaeis guineensis (oil palm), Olea europaea (olive), Oryza sativa (rice), Cucurbita maxima (squash), Hordeum vulgare (barley), and Triticum aestivum (wheat).
[0098] An aspect includes compositions comprising an effective amount of one or more of the dsR As provided in Table 1 for topical treatment of a plant to be treated for, or be protected from, flea beetle infestation. Another aspect includes a recombinant DNA construct encoding at least one strand of at least one the dsRNAs provided in Table 1 for transgenic expression in a plant that has improved resistance to flea beetle infestation, in comparison to a plant not expressing such a construct.
Example 2
[0099] This example illustrates non-limiting embodiments of testing the efficacy of dsRNA sequences and validating the dsR A's utility for suppressing expression of target genes for controlling insect species. More specifically this example illustrates a method comprising contacting an insect, such as a flea beetle adult or larva, with one or more dsRNAs designed to cause stunting or mortality in the insect. Other embodiments include methods where the dsRNA is delivered to the insect by oral delivery (e. g. , on or in a food material ingested by the insect), or through non-oral delivery (e. g. , delivery through the insect's cuticle, or delivery by contacting an egg of the insect).
[00100] In one embodiment, a feeding assay is used to determine efficacy of a dsRNA in causing stunting or mortality in insects, such as flea beetles. To test the efficacy of the dsRNA to kill or stunt flea beetles, a single discriminating dose (for example, 100 nanograms/milliliter) is used to identify dsRNAs with measureable ability to kill or stunt flea beetles at that dose. A negative control dsRNA, such as a dsRNA targetting green fluorescent protein (GFP), is also included in the assay. Each dsRNA is coated evenly onto 1.0 centimeter diameter canola leaf discs and placed in multiwell trays, with 2 male and 2 female adult flea beetles or 4 flea beetle larvae per well. Every 24 hours for a set period (e. g. , 2 weeks), new, freshly-coated leaves are provided. Stunting and mortality are scored periodically (e. g., daily, or every 2 or 3 days).
[00101] The dsRNAs that show efficacy in this single-dose assay are tested further. Using a similar protocol, varying doses of dsRNAs are tested, as described above, to determine the LC50 dose for each of the active dsR As. Bioassays include 12 - 24 insects per dose, performed in triplicate. Stunting and mortality is assessed over a 2 week period, scored on every third day.
[00102] The dsRNA sequences that are confirmed to be effective in suppressing a target gene in a sequence -specific manner are useful for identifying efficacious R A delivery agents and formulations. The insecticidal activity of formulations containing dsRNA can be optimized by various techniques, such as modifying the chemical entities in the formulation or modifying the ratio of the chemical components in the formulation. Non-limiting examples of delivery agents and formulations are provided in Example 5. Example 3
[00103] This example illustrates non-limiting embodiments of methods for validating dsRNA efficacy for suppressing or silencing a target gene in an insect cell or causing stunting or mortality in an insect. More specifically this example illustrates methods for testing dsRNA for efficacy in preventing or treating flea beetle infestations in whole plants.
[00104] Polynucleotides (such as the dsRNA sequences described in Examples 1 and 2) that have been confirmed to be effective in suppressing a target gene in a sequence-specific manner are further evaluated in whole plant assays. In one method, the polynucleotides (e. g., anti-sense RNA, dsRNA) are applied directly to the insect surface (e. g. by spraying or dusting). In another method, the polynucleotides are provided to the insect in an insect diet (e. g. , in a bacterial or plant cell expressing a polynucleotide such as a hairpin form of a dsRNA, or in an artificial bait containing a polynucleotide). Stunting and mortality are scored periodically, as described in Example 2.
[00105] In various methods that are also appropriate for large-scale application (e. g. , to fields of crop plants), the polynucleotide is applied in a foliar application through aerial or terrestrial spraying or dusting or chemigation on the leaf surface to control early season damage from the adult stage of the life cycle, or applied as a seed treatment to control larval or adult stages of the insect life cycle, or applied as a soil in-furrow or drench application to control larval or adult stages of the insect life cycle. An example of a foliar testing regime includes treating the plant immediately after emergence from the ground and evaluating foliar damage caused by adult flea beetles 1 - 2 weeks after plant emergence. For in-furrow or seed treatment similar timing for damage evaluation is followed.
Example 4
[00106] The polynucleotides are generally designed to modulate expression by inducing regulation or suppression of an insect target gene and are designed to have a nucleotide sequence essentially identical or essentially complementary to the nucleotide sequence an insect target gene or cDNA (e. g., SEQ ID NOs:l - 859) or to the sequence of RNA transcribed from an insect target gene, which can be coding sequence or non-coding sequence. This example describes non-limiting techniques useful in the design and selection of polynucleotides to modulate expression of an insect target gene. Selection of Effective Polynucleotides by "Tiling"
[00107] Polynucleotides of use in the compositions and methods disclosed herein need not be of the full length of a target gene, and in many embodiments are of much shorter length in comparison to the target gene. An example of a technique that is useful for selecting effective polynucleotides for insect control is "tiling", or evaluation of polynucleotides corresponding to adjacent or partially overlapping segments of a target gene.
[00108] Effective polynucleotides can be identified by "tiling" gene targets in selected length fragments, e. g. , fragments of 200 - 300 nucleotides in length, with partially overlapping regions, e. g, of about 25 nucleotides, along the length of the target gene. To suppress a single gene, polynucleotides are designed to correspond to (have a nucleotide identity or complementarity with) regions that are unique to the target gene; the selected region of the target gene can include coding sequence or non- coding sequence (e. g. , promoter regions, 3 ' untranslated regions, introns and the like) or a combination of both.
[00109] Where it is of interest to design a polynucleotide effective in suppressing multiple target genes, the multiple target gene sequences are aligned and polynucleotides designed to correspond to regions with high sequence homology in common among the multiple targets. Conversely, where it is of interest to design a polynucleotide effective in selectively suppressing one among multiple target sequences, the multiple target gene sequences are aligned and polynucleotides designed to correspond to regions with no or low sequence homology in common among the multiple targets.
[00110] In a non-limiting example, polynucleotides (e.g, anti -sense single -stranded RNAs, dsRNAs, anti-sense single-stranded DNAs, dsDNAs) capable of inducing suppression are designed for each of the target genes listed in Table 1 as follows. Multiple polynucleotides, each of 200 - 300 nucleotides in length and comprising a sequence complementary to a fragment of a target gene having a sequence selected from SEQ ID NOs:l - 859 are designed so that each polynucleotide's sequence overlaps about 25 nucleotides of the next adjacent polynucleotide's sequence, in such a way that the multiple polynucleotides in combination cover the full length of the target gene. (Similarly, double- stranded polynucleotides can be designed by providing pairs of sense and anti -sense polynucleotides, each pair of polynucleotides overlapping the next adjacent pair of polynucleotides.)
[00111] The polynucleotides are tested by any convenient means for efficacy in silencing the insect target gene. Examples of a suitable test include the bioassays described herein in the working Examples. Another test involves the topical application of the polynucleotides either directly to individual insects or to the surface of a plant to be protected from an insect infestation. One desired result of treatment with a polynucleotide as disclosed herein is prevention or control of an insect infestation, e. g. , by inducing in an insect a physiological or behavioural change such as, but not limited to, growth stunting, increased mortality, decrease in reproductive capacity, decrease in or cessation of feeding behavior or movement, or decrease in or cessation of metamorphosis stage development. Another desired result of treatment with a polynucleotide as disclosed herein is provision of a plant that exhibits improved resistance to an insect infestation. [00112] The tiling procedure can be repeated, if desired. A polynucleotide found to provide desired activity can itself be subjected to a tiling procedure. For example, multiple overlapping polynucleotides are designed, each of 50 - 60 nucleotides in length and comprising a sequence complementary to the fragment of a target gene having a sequence selected from SEQ ID NOs:l - 859 for which a single polynucleotide of 300 nucleotides was found to be effective. Additional rounds of tiling analysis can be carried out, where polynucleotides as short as 18, 19, 20, or 21 nucleotides are tested.
[00113] Effective polynucleotides of any size can be used, alone or in combination, in the various methods disclosed herein. In some embodiments, a single polynucleotide is used to make a composition (e. g. , a composition for topical application, or a recombinant DNA construct useful for making a transgenic plant). In other embodiments, a mixture or pool of different polynucleotides is used; in such cases the polynucleotides can be for a single target gene or for multiple target genes. In some embodiments, a polynucleotide is designed to target different regions of the target gene, e. g. , an insecticidal polynucleotide can comprise multiple segments that correspond to different exon regions of the target gene, and "spacer" nucleotides which do not correspond to a target gene can optionally be used in between or adjacent to the segments.
Thermodynamic Considerations in Selecting Insecticidal Polynucleotides
[00114] Polynucleotides can be designed or their sequence optimised using thermodynamic considerations. For example, insecticidal polynucleotides can be selected based on the thermodynamics controlling hybridization between one nucleic acid strand (e. g. , a polynucleotide or an individual siRNA) and another (e. g. , a target gene transcript)
[00115] Methods and algorithms to predict nucleotide sequences that are likely to be effective at RNAi-mediated silencing of a target gene are known in the art. Non-limiting examples of such methods and algorithms include "i-score", described by Ichihara et al. (2007) Nucleic Acids Res. , 35(18): 123e; "Oligowalk", publicly available at rna.urmc.rochester.edu/servers/oligowalk and described by Lu et al. (2008) Nucleic Acids Res. , 36:W104-108; and "Reynolds score", described by Khovorova et al. (2004) Nature Biotechnol , 22:326-330.
Permitted mismatches
[00116] By "essentially identical" or "essentially complementary" is meant that the polynucleotide (or at least one strand of a double-stranded polynucleotide) has sufficient identity or complementarity to the target gene or to the RNA transcribed from a target gene (e. g. , the transcript) to suppress expression of a target gene (e. g. , to effect a reduction in levels or activity of the target gene transcript and/or encoded protein). Polynucleotides need not have 100 percent identity or
complementarity to a target gene or to the RNA transcribed from a target gene to suppress expression of the target gene (e. g. , to effect a reduction in levels or activity of the target gene transcript or encoded protein, or to provide control of an insect species). In some embodiments, the polynucleotide or a portion thereof is designed to be essentially identical to, or essentially complementary to, a sequence of at least 18 or 19 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In some embodiments, the polynucleotide or a portion thereof is designed to be exactly identical to, or exactly complementary to, a sequence of 21 contiguous nucleotides in either the target gene or the RNA transcribed from the target gene. In certain embodiments, an "essentially identical"
polynucleotide has 100 percent sequence identity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence identity when compared to the sequence of 18 or more contiguous nucleotides of the target gene or an RNA transcribed from the target gene. In certain embodiments, an "essentially complementary" polynucleotide has 100 percent sequence
complementarity or at least about 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent sequence complementarity to 18 or more contiguous nucleotides of the target gene or RNA transcribed from the target gene .
[00117] Polynucleotides comprising mismatches to the target gene or transcript can be used in certain embodiments of the compositions and methods disclosed herein. In some embodiments, the polynucleotide comprises at least 18 or at least 19 contiguous nucleotides that are essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript. In certain embodiments, a polynucleotide of 19 contiguous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 20 or more nucleotides that comprises a contiguous 19 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1 or 2 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 21 continuous nucleotides that is essentially identical or essentially complementary to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript. In certain embodiments, a polynucleotide of 22 or more nucleotides that contains a contiguous 21 nucleotide span of identity or complementarity to a segment of equivalent length in the target gene or target gene's transcript can have 1, 2, or 3 mismatches to the target gene or transcript.
[00118] In designing polynucleotides with mismatches to a target gene or to an RNA transcribed from the target gene, mismatches of certain types and at certain positions that are more likely to be tolerated can be used. In certain embodiments, mismatches formed between adenine and cytosine or guanosine and uracil residues are used as described by Du et al. (2005) Nucleic Acids Res. , 33 : 1671-1677. In some embodiments, mismatches in 19 base-pair overlap regions are located at the low tolerance positions 5, 7, 8 or 1 1 (from the 5 ' end of a 19-nucleotide target), at medium tolerance positions 3, 4, and 12-17(from the 5 ' end of a 19-nucleotide target), and/or at the high tolerance positions at either end of the region of complementarity, e. g , positions 1, 2, 18, and 19 (from the 5' end of a 19-nucleotide target) as described by Du et al. (2005) Nucleic Acids Res. , 33 : 1671-1677.
Tolerated mismatches can be empirically determined in routine assays such as those described herein in the working Examples. [00119] In some embodiments, the polynucleotides comprise additional nucleotides (e.g., for reasons of stability or for convenience in cloning or synthesis). In one embodiment, the polynucleotide is a single -stranded RNA comprising an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 and further comprising an additional 5' G or an additional 3' C or both, adjacent to the segment. In one embodiment, the polynucleotide is a dsR A comprising an RNA strand with a segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 and further comprising an additional 5' G or an additional 3' C or both, adjacent to the segment. In another embodiment, the polynucleotide is a double -stranded RNA comprising additional nucleotides to form an overhang, for example, a dsRNA comprising 2 deoxyribonucleotides to form a 3' overhang.
Embedding Active Insecticidal Polynucleotides in Neutral Sequence
[00120] In an embodiment, a a polynucleotide with a sequence complementary to the target gene and which is responsible for an observed suppression of the target gene is embedded in "neutral" sequence, e. g. , inserted into additional nucleotides that have no sequence identity or complementarity to the target gene. Neutral sequence can be desirable, e. g. , to increase the overall length of a polynucleotide. For example, it can be desirable for a polynucleotide to be of a particular size for reasons of stability, cost-effectiveness in manufacturing, or biological activity.
[00121] It has been reported that in another coleopteran species, Diabrotica virgifera, dsRNAs greater than or equal to approximately 60 base-pairs (bp) are required for biological activity in artificial diet bioassays; see Bolognesi et al. (2012) PLoS ONE 7(10): e47534.
doi: 10.1371/journal.pone.0047534. Thus, in one embodiment, a 21-base-pair dsRNA corresponding to a target gene in Table 1 and found to provide control of an insect infestation is embedded in neutral sequence of an additional 39 base pairs, thus forming a polynucleotide of about 60 base pairs. In another embodiment, a single 21-base-pair polynucleotide is found to be efficacious when embedded in larger sections of neutral sequence, e. g. , where the total polynucleotide length is from about 60 to about 300 base pairs. In another embodiment, at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 is embedded in larger sections of neutral sequence to provide an efficacious insecticidal polynucleotide. In another embodiment, segments from multiple sequences selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975 are embedded in larger sections of neutral sequence to provide an efficacious insecticidal polynucleotide.
[00122] It is anticipated that the combination of certain recombinant polynucleotides disclosed herein (e. g., single-strand RNA, dsRNA, single-strand DNA, or dsDNAcomprising a sequence selected from SEQ ID NOs:860 - 1718 and 1722 - 1975, or active fragments thereof) with one or more non-polynucleotide pesticidal agents will result in an improvement in prevention or control of insect infestations, when compared to the effect obtained with the polynucleotide alone or the non- polynucleotide pesticidal agent alone. Routine insect bioassays such as the bioassays described herein in the working Examples are useful for defining dose-responses for larval mortality or growth inhibition using combinations of the polynucleotides disclosed herein and one or more non-polynucleotide pesticidal agents (e. g. , a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, aXenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus
laterosporous insecticidal protein, and a Bacillus sphaericus insecticidal protein). One of skill in the art can test combinations of polynucleotides and non-polynucleotide pesticidal agents in routine bioassays to identify combinations of bioactives that are effective for use in protecting plants from insect infestations. Example 5
[00123] This example illustrates non-limiting embodiments of methods of testing the efficacy of insecticidal polynucleotides in flea beetles. More specifically this example illustrates a method including oral delivery of polynucleotides to flea beetles, resulting in stunting or mortality in the flea beetles.
[00124] P. cruciferae were collected from a canola field where no pesticides had been applied in the previous 3 months. Three dsRNAs (SEQ ID NOs:1169, 1193, and 1392) targeting Phyllotreta genes and one negative control dsRNA targeting GFP were tested on groups of 30 P.
cruciferae. The dsRNAs were resuspended in water and applied to 6 millimeter leaf discs (55 ± 6 milligrams each) at a discriminating dose of 50 nanograms dsR A/milligram leaf tissue, which were fed to groups of 5 flea beetles. Leaf discs with freshly applied dsRNA were replaced every other day, and the number of surviving individuals was recorded over a 2-week period. A low non-specific mortality rate was observed in the negative-control insect groups (3 out of 30 insects dying over 2 weeks, or 10% non-specific mortality). Mortality was observed beginning at day 4 and continuing through the 2 week period. Specific mortality was observed for all dsRNA treatments (Table 2).
Correcting for non-specific mortality (subtracting non-specific mortality rate of 3 insects per group for corrected N= 27), the percent mortality observed at the end of the 2-week period was 85% (SEQ ID NO:1169), 0.70% (SEQ ID NO:1193), and 0.63% (SEQ ID NO:1392). These results demonstrated the efficacy of the dsRNAs in causing mortality in flea beetles when provided in the flea beetles' diet. Table 2: Cumulative number of dead P. cruciferae (N= 30)
Figure imgf000057_0001
[00125] In a second series of experiments carried out in a similar manner, several dsR As were tested at a discriminating dose of 50 nanograms dsRNA/milligram leaf tissue on P. cruciferae; two lower doses (15 nanograms dsRNA/milligram leaf tissue and 2 nanograms dsRNA/milligram leaf tissue) were also tested. Ten beetles were tested at each dose. The negative control (five replicates) used was a dsR A targetting the bacterial gene uidA encoding fteto-glucuronidase (NCBI accession number NC_000913.3). Leaf discs with freshly applied dsRNA were replaced every other day, and mortality was recorded over a 12 day period. The overall mortality rate for the negative controls was -4% (likely due to handling injuries) over the 12-day observation period. The observed cumulative mortality (N = 10) following 12 days exposure to the dsRNAs are provided in Table 3; the negative control mortality values are given as an average (N= 5). Eight of the dsRNAs (indicated by a mortality rating of +++) caused 90 - 100% mortality at the highest dose and were still highly effective (80% or higher mortality) at the lowest dose tested. Some dsRNAs (indicated by a mortality rating of ++) induced mortality in a proportion of the insects at the highest dose, but were less effective at lower doses (<20% mortality).
Table 3
Figure imgf000058_0001
Figure imgf000059_0001
*+++ rating indicates high (>80%) mortalities for all three doses; ++ rating indicates high mortalities for the highest dose, and within 40 to 70% mortality with the lower two doses.
** sequences are provided for the anti -sense strand of the dsRNA in 5' to 3' direction.
[00126] Other techniques for delivering these or similar insecticidal polynucleotides are contemplated and include applying the polynucleotides directly to the insect surface (e. g. by spraying or dusting), or providing the polynucleotide sto the insect in a diet or bait (e. g., in a bacterial or plant cell expressing a dsRNA, or in an artificial bait containing the dsRNA). In an embodiment, a hairpin version of the Phyllotreta insecticidal polynucleotide with the sequence SEQ ID NO: 1169 is designed; this hairpin version is encoded by the DNA sequence SEQ ID NO:1722, which contains, in 5' to 3' order, anti-sense sequence (nucleotide positions 1 - 267), loop sequence (nucleotide positions 268 - 373) which does not contain matches to Phyllotreta sequences, and sense sequence (nucleotide positions 374 - 640). This DNA sequence is expressed as a single-stranded RNA transcript, where the anti-sense and sense regions anneal to form the double-stranded "stem" region of the hairpin. The construct is expressed in a bacterium, such as E. coli; the resulting dsRNA hairpin produced in the bacterium is provided to flea beetles as a crude or purified fermentation product, or in the form of the bacterial cells. Similar constructs are designed encoding dsRNAs having modified stem-loops, such as "stabilized anti-sense" or "stabilized sense" versions, which contain stabilized loops formed by an extended anti-sense or sense sequence, respectively, of sequence corresponding to the intended target gene. Example 6
[00127] This example discloses embodiments related to polynucleotide molecules having a nucleotide sequence containing specific modifications such as nucleotide substitutions. Embodiments of such modifications include modified polynucleotides that provide improved sequence discrimination between the intended target gene of the insect pest of interest, and genetic sequences of other, non- target species.
[00128] Selected dsRNAs identified in Table 1 were screened for unintended sequence matches to a sequence of at least 19 contiguous nucleotides identified in a non -target gene or a non- target organism (NTO, e. g., Apis mellifera, Bombus impatiens and B. terrestris; Coleomegilla spp.; Danaus plexippus; Homo sapiens; Megachile rotundata; Mus musculus; and Brassica rapa).
Nucleotide changes are made in an original polynucleotide sequence to eliminate contiguous sequence matches to a non-target gene or non-target organism. Examples of such modified polynucleotide sequences are provided by SEQ ID NO:1723, which corresponds to SEQ ID NO:1393 (which targets the same flea beetle gene as does the insecticidal polynucleotide of SEQ ID NO: 1392), and SEQ ID NO:1724, which corresponds to SEQ ID NO:1169.
Example 7
[00129] This example discloses embodiments related to polynucleotide molecules having a nucleotide sequence for silencing a target gene in more than one species. Embodiments include dsRNA sequences of at least 21 contiguous nucleotides identified as having 100% complementarity or identity to more than one ortholog of a target gene.
[00130] Table 4 provides a list of sequences, each at least 21 contiguous nucleotides in length and identified by the sequence's coordinates in a dsRNA for one flea beetle species, wherein the identical sequence is also found in a dsRNA for a different flea beetle species. These sequences are useful to design insecticidal polynucleotides against multiple species in which the target sequence co- occurs. For example, SEQ ID NO:1186 (targetting a Phyllotreta cruciferae COPI alpha target gene, SEQ ID NO:327) contains five sequences of at least 21 contiguous nucleotides at positions 1 - 71, 88 - 116, 136 - 209, 238 - 266, and 274 - 296, all of which match a sequence in SEQ ID NOs:882 and 888 (targetting a. Phyllotreta atra COPI alpha target genes, SEQ ID NOs:23 and 29, respectively); these five sequences are therefore useful in targeting a gene in the two Phyllotreta species.
Table 4
Figure imgf000060_0001
1039 - 1043, 1045, 1594, 1597,
1186 136 209 882, 888 1334 109 185 1598
1039 - 1043, 1045, 1594, 1597,
1186 238 266 882, 888 1334 187 254 1598
1039 - 1043, 1045, 1594, 1597,
1186 274 296 882, 888 1334 271 302 1598
1039 - 1043, 1045, 1594, 1597,
1191 51 96 900 - 908, 910 1335 2 47 1598
1039 - 1043, 1045, 1594, 1597,
1191 137 177 900 - 908, 910 1335 49 116 1598
1039 - 1043, 1045, 1594, 1597,
1191 179 216 900 - 908, 910 1335 133 164 1598
1039 - 1043, 1045, 1594, 1597,
1191 218 255 900 - 908, 910 1335 229 251 1598
1039 - 1043, 1045, 1594, 1597,
1191 257 342 900 - 908, 910 1336 34 90 1598
1039 - 1043, 1045, 1594, 1597,
1192 2 25 900 - 908, 910 1336 101 177 1598
1039 - 1043, 1045, 1594, 1597,
1192 66 106 900 - 908, 910 1336 179 246 1598
1039 - 1043, 1045, 1594, 1597,
1192 108 145 900 - 908, 910 1336 263 294 1598
1039 - 1043, 1045, 1594, 1597,
1192 147 184 900 - 908, 910 1338 40 96 1598
1039 - 1043, 1045, 1594, 1597,
1192 186 271 900 - 908, 910 1338 107 183 1598
1039 - 1043, 1045, 1594, 1597,
1192 273 298 900 - 908, 910 1338 185 252 1598
1039 - 1043, 1045, 1594, 1597,
1192 300 330 900 - 908, 910 1338 269 300 1598
1039 - 1043, 1045, 1594, 1597,
1191 58 96 900 - 908, 910 1328 35 59 1598
1039 - 1043, 1045, 1594, 1597,
1192 300 336 900 - 908, 910 1331 60 84 1598
1039 - 1043, 1045, 1594, 1597,
1191 44 96 900 - 908, 910 1332 1 26 1598
1039 - 1043, 1045, 1594, 1597,
1191 179 200 900 - 908, 910 1333 36 60 1598
1039 - 1043, 1045, 1594, 1597,
1192 108 129 900 - 908, 910 1334 74 98 1598
1039 - 1043, 1045, 1594, 1597,
1191 218 242 900 - 908, 910 1336 66 90 1598
1039 - 1043, 1045, 1594, 1597,
1192 147 171 900 - 908, 910 1338 72 96 1598
1039 - 1043, 1045, 1594, 1597,
1191 257 277 900 - 908, 910 1331 1 26 1598
1039 - 1043, 1045, 1594, 1597,
1192 186 206 900 - 908, 910 1334 2 40 1598
1039 - 1043, 1045, 1594, 1597,
1191 257 334 900 - 908, 910 1336 2 32 1598
1039 - 1043, 1045, 1594, 1597,
1192 186 263 900 - 908, 910 1338 2 38 1598
1039 - 1043, 1045, 1594, 1597,
1191 257 320 900 - 908, 910 1328 36 59 1598
1039 - 1043, 1045, 1594, 1597,
1192 186 249 900 - 908, 910 1331 61 84 1598
1039 - 1043, 1045, 1594, 1597,
1193 15 101 911, 915, 916, 919 1332 3 26 1598
1039 - 1043, 1045, 1594, 1597,
1194 6 92 911, 915, 916, 919 1333 37 60 1598
1195 2 107 911, 915, 916, 919 1334 75 98 1039 - 1043, 1045, 1594, 1597, 1598
1039 - 1043, 1045, 1594, 1597,
1195 121 207 911,915,916, 919 1336 67 90 1598
1039 - 1043, 1045, 1594, 1597,
1196 2 122 911,915,916, 919 1338 73 96 1598
1196 136 222 911,915,916, 919 1329 172 191 1039-1043 1045
1197 2 20 911,915,916, 919 1329 265 296 1039-1043 1045
1197 22 42 911,915,916, 919 1329 298 317 1039-1043 1045
1197 44 180 911,915,916, 919 1329 319 343 1039-1043 1045
1197 194 280 911,915,916, 919 1330 181 200 1039-1043 1045
1198 18 38 911,915,916, 919 1330 274 305 1039-1043 1045
1198 40 176 911,915,916, 919 1330 307 326 1039-1043 1045
1198 190 276 911,915,916, 919 1330 328 352 1039-1043 1045
1039 - 1043, 1045, 1594, 1597,
1199 5 25 911,915,916, 919 1335 283 302 1598
1199 27 163 911,915,916, 919 1337 1 19 1043, 1045
1199 177 263 911,915,916, 919 1337 93 124 1043, 1045
1200 16 152 911,915,916, 919 1337 126 145 1043, 1045
1200 166 252 911,915,916, 919 1337 147 171 1043, 1045
1201 19 105 911,915,916, 919 1329 319 352 1039-1043 1045
1202 2 119 911,915,916, 919 1337 147 180 1043, 1045
1202 133 219 911,915,916, 919 1386 2 38 1054 - 1057, 1060, 1496, 1498
1203 14 100 911,915,916, 919 1386 40 59 1054 - 1057, 1060, 1496, 1498
1193 123 189 911,915,916, 919 1386 70 95 1054 - 1057, 1060, 1496, 1498
1194 114 180 911,915,916, 919 1386 103 161 1054 - 1057, 1060, 1496, 1498
1195 229 295 911,915,916, 919 1386 196 262 1054 - 1057, 1060, 1496, 1498
1196 244 310 911,915,916, 919 1388 2 51 1054 - 1057, 1060, 1496, 1498
1197 302 350 911,915,916, 919 1388 53 72 1054 - 1057, 1060, 1496, 1498
1198 298 350 911,915,916, 919 1388 83 108 1054 - 1057, 1060, 1496, 1498
1199 285 352 911,915,916, 919 1388 116 174 1054 - 1057, 1060, 1496, 1498
1200 274 340 911,915,916, 919 1388 209 275 1054 - 1057, 1060, 1496, 1498
1054 - 1057, 1060, 1492, 1496
1201 127 193 911,915,916, 919 1390 88 119 -1498
1054 - 1057, 1060, 1492, 1496
1202 241 307 911,915,916, 919 1390 145 194 -1498
1054 - 1057, 1060, 1492, 1496
1203 122 188 911,915,916, 919 1390 196 215 -1498
1054 - 1057, 1060, 1492, 1496
1193 123 196 911,915,916, 919 1390 226 251 -1498
1054 - 1057, 1060, 1492, 1496
1193 198 230 911,915,916, 919 1390 259 317 -1498
1054 - 1057, 1060, 1492, 1496
1194 114 187 911,915,916, 919 1391 19 50 -1498
1054 - 1057, 1060, 1492, 1496
1194 189 221 911,915,916, 919 1391 76 125 -1498
1054 - 1057, 1060, 1492, 1496
1195 229 302 911,915,916, 919 1391 127 146 -1498
1054 - 1057, 1060, 1492, 1496
1195 304 336 911,915,916, 919 1391 157 182 -1498
1054 - 1057, 1060, 1492, 1496
1196 1 122 911,915,916, 919 1391 190 248 -1498
1054 - 1057, 1060, 1492, 1496
1196 244 317 911,915,916, 919 1391 283 349 -1498
1055, 1058, 1059, 1061 - 1063,
1196 319 351 911,915,916, 919 1384 20 39 1500
1055, 1058, 1059, 1061 - 1063,
1197 60 180 911,915,916, 919 1384 62 84 1500
1055, 1058, 1059, 1061 - 1063,
1198 56 176 911,915,916, 919 1384 134 204 1500
1199 43 163 911,915,916, 919 1384 206 249 1055, 1058, 1059, 1061 - 1063, 1500
1055, 1058, 1059, 1061 - 1063,
1199 285 351 911, 915, 916, 919 1384 251 270 1500
1055, 1058, 1059, 1061 - 1063,
1200 32 152 911, 915, 916, 919 1384 284 305 1500
1055, 1058, 1059, 1061 - 1063,
1200 274 347 911, 915, 916, 919 1385 21 40 1500
1055, 1058, 1059, 1061 - 1063,
1201 127 200 911, 915, 916, 919 1385 63 85 1500
1055, 1058, 1059, 1061 - 1063,
1201 202 234 911, 915, 916, 919 1385 135 205 1500
1055, 1058, 1059, 1061 - 1063,
1202 241 314 911, 915, 916, 919 1385 207 250 1500
1055, 1058, 1059, 1061 - 1063,
1202 316 348 911, 915, 916, 919 1385 252 271 1500
1055, 1058, 1059, 1061 - 1063,
1203 122 195 911, 915, 916, 919 1385 285 306 1500
1055, 1058, 1059, 1061 - 1063,
1203 197 229 911, 915, 916, 919 1387 2 21 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1204 6 25 920, 921, 922, 923, 924 1387 23 42 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1204 27 49 920, 921, 922, 923, 924 1387 56 77 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1204 51 118 920, 921, 922, 923, 924 1389 1 55 1500
1055, 1058, 1059, 1061 - 1063,
1204 186 214 920, 921, 922, 923, 924 1389 57 100 1500
1055, 1058, 1059, 1061 - 1063,
1204 219 268 920, 921, 922, 923, 924 1389 102 121 1500
1055, 1058, 1059, 1061 - 1063,
1204 270 295 920, 921, 922, 923, 924 1389 135 156 1500
1204 306 351 920, 921, 922, 923, 924 1386 196 255 1054 - 1057, 1060, 1496, 1498
1205 180 199 920, 921, 922, 923, 924 1388 209 268 1054 - 1057, 1060, 1496, 1498
1054 - 1057, 1060, 1492, 1496
1205 201 223 920, 921, 922, 923, 924 1391 283 342 - 1498
1055, 1058, 1059, 1061 - 1063,
1205 225 292 920, 921, 922, 923, 924 1387 257 321 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1206 10 29 920, 921, 922, 923, 924 1387 332 351 1500, 1057
1054 - 1057, 1060, 1492, 1496
1206 31 53 920, 921, 922, 923, 924 1390 25 56 - 1498
1055, 1058, 1059, 1061 - 1063,
1206 55 122 920, 921, 922, 923, 924 1384 284 321 1500
1055, 1058, 1059, 1061 - 1063,
1206 190 218 920, 921, 922, 923, 924 1385 285 322 1500
1055, 1058, 1059, 1061 - 1063,
1206 223 272 920, 921, 922, 923, 924 1387 56 93 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1206 274 299 920, 921, 922, 923, 924 1389 135 172 1500
1206 310 351 920, 921, 922, 923, 924 1386 196 332 1054 - 1057, 1060, 1496, 1498
1207 11 33 920, 921, 922, 923, 924 1388 209 345 1054 - 1057, 1060, 1496, 1498
1054 - 1057, 1060, 1492, 1496
1207 35 102 920, 921, 922, 923, 924 1391 283 352 - 1498
1055, 1058, 1059, 1061 - 1063,
1207 170 198 920, 921, 922, 923, 924 1384 323 348 1500
1055, 1058, 1059, 1061 - 1063,
1207 203 252 920, 921, 922, 923, 924 1385 324 349 1500
1055, 1058, 1059, 1061 - 1063,
1207 254 279 920, 921, 922, 923, 924 1387 95 120 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1207 290 335 920, 921, 922, 923, 924 1387 128 153 1500, 1057 1055, 1058, 1059, 1061 - 1063,
1208 18 40 920, 921, 922, 923, 924 1387 161 186 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1208 42 109 920, 921, 922, 923, 924 1387 191 210 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1208 177 205 920, 921, 922, 923, 924 1389 174 199 1500
1055, 1058, 1059, 1061 - 1063,
1208 210 259 920, 921, 922, 923, 924 1389 207 232 1500
1055, 1058, 1059, 1061 - 1063,
1208 261 286 920, 921, 922, 923, 924 1389 240 265 1500
1055, 1058, 1059, 1061 - 1063,
1208 297 342 920, 921, 922, 923, 924 1389 270 289 1500
1209 101 120 920, 921, 922, 923, 924 1283 177 208 1064 - 1070
1209 122 144 920, 921, 922, 923, 924 1284 130 161 1064 - 1070
1209 146 213 920, 921, 922, 923, 924 1284 301 351 1064 - 1070
1209 281 309 920, 921, 922, 923, 924 1286 210 241 1064 - 1070
1209 314 351 920, 921, 922, 923, 924 1287 126 157 1064 - 1070
1205 2 91 920, 921, 922, 923, 924 1287 297 351 1064 - 1070
1205 114 142 920, 921, 922, 923, 924 1288 326 351 1064 - 1070, 1071
1205 144 178 920, 921, 922, 923, 924 1289 41 72 1064 - 1070
1209 35 63 920, 921, 922, 923, 924 1289 212 288 1064 - 1070
1209 65 99 920, 921, 922, 923, 924 1289 320 351 1064 - 1070
1204 306 347 920, 921, 922, 923, 924 1290 178 209 1064 - 1070
1207 290 331 920, 921, 922, 923, 924 1291 228 259 1064 - 1070, 1071
1208 297 338 920, 921, 922, 923, 924 1292 43 74 1064 - 1070
1207 290 352 920, 921, 922, 923, 924 1292 214 290 1064 - 1070
1208 297 351 920, 921, 922, 923, 924 1292 322 351 1064 - 1070
1205 18 91 920, 921, 922, 923, 924 1293 330 351 1064 - 1070, 1071
1224 1 43 947 - 949, 951 - 956 1377 37 68 1064 - 1070
1224 57 95 947 - 949, 951 - 956 1377 208 284 1064 - 1070
1224 97 145 947 - 949, 951 - 956 1377 316 348 1064 - 1070
1224 147 175 947 - 949, 951 - 956 1289 212 274 1064 - 1070
1224 177 208 947 - 949, 951 - 956 1292 214 276 1064 - 1070
1224 210 262 947 - 949, 951 - 956 1377 208 270 1064 - 1070
1224 285 350 947 - 949, 951 - 956 1285 123 148 1067, 1069, 1071
1225 150 192 947, 949, 951 - 956 1288 107 132 1064 - 1070, 1071
1225 206 244 947, 949, 951 - 956 1291 9 34 1064 - 1070, 1071
1225 246 294 947, 949, 951 - 956 1293 111 136 1064 - 1070, 1071
1225 296 324 947, 949, 951 - 956 1287 297 347 1064 - 1070
1225 326 351 947, 949, 951 - 956 1289 212 262 1064 - 1070
1226 258 300 947, 949, 951 - 955 1292 214 264 1064 - 1070
1226 314 351 947, 949, 951 - 955 1377 208 258 1064 - 1070
1227 253 295 947, 949, 951 - 955 1287 297 352 1064 - 1070
1227 309 347 947, 949, 951 - 955 1289 212 266 1064 - 1070
1228 206 248 947, 949, 951 - 956 1292 214 268 1064 - 1070
1228 262 300 947, 949, 951 - 956 1377 208 262 1064 - 1070
1228 302 350 947, 949, 951 - 956 1285 18 61 1067, 1069, 1071
1229 171 213 947, 949, 951 - 956 1288 2 45 1064 - 1070, 1071
1229 227 265 947, 949, 951 - 956 1293 6 49 1064 - 1070, 1071
1079 - 1082, 1152 - 1154,
1229 267 315 947, 949, 951 - 956 1357 1 40 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1229 317 345 947, 949, 951 - 956 1357 63 85 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1230 252 294 947, 949, 951 - 955 1357 102 157 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1230 308 346 947, 949, 951 - 955 1357 159 189 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1231 191 233 947, 949, 951 - 956 1357 191 322 1455, 1457, 1458
1231 247 285 947, 949, 951 - 956 1358 25 47 1079 - 1082, 1152 - 1154, 1231 287 335 947, 949, 951 - 956
1232 196 238 947, 949, 951 - 956
1232 252 290 947, 949, 951 - 956
1232 292 340 947, 949, 951 - 956
1233 209 251 947, 949, 951 - 956
1233 265 303 947, 949, 951 - 956
1233 305 351 947, 949, 951 - 956
1224 177 202 947 - 949, 951 - 956
1225 1 72 947, 949, 951 - 956
1225 74 135 947, 949, 951 - 956
1225 137 192 947, 949, 951 - 956
1226 110 180 947, 949, 951 - 955
1226 182 243 947, 949, 951 - 955
1226 245 300 947, 949, 951 - 955
1227 105 175 947, 949, 951 - 955
1227 177 238 947, 949, 951 - 955
1227 240 295 947, 949, 951 - 955
1228 58 128 947, 949, 951 - 956
1228 130 191 947, 949, 951 - 956
1228 193 248 947, 949, 951 - 956
1229 23 93 947, 949, 951 - 956
1229 95 156 947, 949, 951 - 956
1229 158 213 947, 949, 951 - 956
1230 104 174 947, 949, 951 - 955
1230 176 237 947, 949, 951 - 955
1230 239 294 947, 949, 951 - 955
1231 43 113 947, 949, 951 - 956
1231 115 176 947, 949, 951 - 956
1231 178 233 947, 949, 951 - 956
1232 48 118 947, 949, 951 - 956
Figure imgf000065_0001
1079-1082,1152-1154,
1232 120 181 947, 949, 951 - 956 1365 163 294 1455, 1457, 1458
1079-1082,1152-1154,
1232 183 238 947, 949, 951 - 956 1366 2 26 1455, 1457, 1458
1079-1082,1152-1154,
1233 61 131 947, 949, 951 - 956 1366 28 58 1455, 1457, 1458
1079-1082,1152-1154,
1233 133 194 947, 949, 951 - 956 1366 60 191 1455, 1457, 1458
1079, 1080, 1081, 1082, 1152,
1233 196 251 947, 949, 951 - 956 1367 2 116 1153, 1154
1079-1082,1152-1154,
1224 227 262 947 - 949, 951 - 956 1358 322 345 1455, 1457, 1458
1079-1082,1152-1154,
1224 57 94 947 - 949, 951 - 956 1359 328 350 1455, 1457, 1458
1079-1082,1152-1154,
1225 2 72 947, 949, 951 - 956 1362 328 351 1455, 1457, 1458
1079-1082,1152-1154,
1225 206 243 947, 949, 951 - 956 1363 330 352 1455, 1457, 1458
1079-1082,1152-1154,
1226 1 96 947, 949, 951 - 955 1364 262 285 1455, 1457, 1458
1079-1082,1152-1154,
1226 98 180 947, 949, 951 - 955 1365 332 351 1455, 1457, 1458
1079-1082,1152-1154,
1227 2 91 947, 949, 951 - 955 1366 229 252 1455, 1457, 1458
1227 93 175 947, 949, 951 - 955 1367 154 177 1079-1082,1152-1154
1079-1082,1152-1154,
1227 309 346 947, 949, 951 - 955 1358 322 344 1455, 1457, 1458
1079-1082,1152-1154,
1228 2 44 947, 949, 951 - 956 1362 328 350 1455, 1457, 1458
1079-1082,1152-1154,
1228 46 128 947, 949, 951 - 956 1364 262 284 1455, 1457, 1458
1079-1082,1152-1154,
1228 262 299 947, 949, 951 - 956 1366 229 251 1455, 1457, 1458
1229 11 93 947, 949, 951 - 956 1367 154 176 1079-1082,1152-1154
1229 227 264 947, 949, 951 - 956 1368 126 145 1083, 1087
1230 2 90 947, 949, 951 - 955 1368 153 177 1083, 1087
1230 92 174 947, 949, 951 - 955 1368 186 208 1083, 1087
1230 308 345 947, 949, 951 - 955 1368 224 253 1083, 1087
1231 2 29 947, 949, 951 - 956 1369 161 180 1083, 1084, 1089
1231 31 113 947, 949, 951 - 956 1370 124 143 1083, 1087
1231 247 284 947, 949, 951 - 956 1370 151 175 1083, 1087
1232 2 34 947, 949, 951 - 956 1370 184 206 1083, 1087
1232 36 118 947, 949, 951 - 956 1370 222 251 1083, 1087
1232 252 289 947, 949, 951 - 956 1369 278 303 1083, 1084, 1089
1233 2 47 947, 949, 951 - 956 1369 182 201 1083, 1084, 1089
1233 49 131 947, 949, 951 - 956 1371 1 71 1093 - 1095, 1098, 1100 -1103
1233 265 302 947, 949, 951 - 956 1371 82 122 1093 - 1095, 1098, 1100 -1103
1226 7 96 947, 949, 951 - 955 1371 124 180 1093 - 1095, 1098, 1100 -1103
1227 1 91 947, 949, 951 - 955 1371 182 244 1093 - 1095, 1098, 1100 -1103
1226 8 96 947, 949, 951 - 955 1372 16 56 1093 - 1095, 1098, 1100 -1103
1227 3 91 947, 949, 951 - 955 1372 58 114 1093 - 1095, 1098, 1100 -1103
1230 1 90 947, 949, 951 - 955 1372 116 178 1093 - 1095, 1098, 1100 -1103
1226 54 96 947, 949, 951 - 955 1373 2 61 1093 - 1095, 1098, 1100 -1103
1227 49 91 947, 949, 951 - 955 1373 72 112 1093 - 1095, 1098, 1100 -1103
1228 1 44 947, 949, 951 - 956 1373 114 170 1093 - 1095, 1098, 1100 -1103
1230 48 90 947, 949, 951 - 955 1373 172 234 1093 - 1095, 1098, 1100 -1103
1233 5 47 947, 949, 951 - 956 1374 1 68 1093 - 1095, 1098, 1100 -1103
1224 125 145 947 - 949, 951 - 956 1374 79 119 1093 - 1095, 1098, 1100 -1103
1225 274 294 947, 949, 951 - 956 1374 121 177 1093 - 1095, 1098, 1100 -1103
1228 330 350 947, 949, 951 - 956 1374 179 241 1093 - 1095, 1098, 1100 -1103 1229 295 315 947, 949, 951 - 956 1375 2 41 1093 - 1095, 1098, 1100 -1103
1231 315 335 947, 949, 951 - 956 1375 52 92 1093 - 1095, 1098, 1100 -1103
1232 320 340 947, 949, 951 - 956 1375 94 150 1093 - 1095, 1098, 1100 -1103
1233 333 351 947, 949, 951 - 956 1375 152 214 1093 - 1095, 1098, 1100 -1103
1234 50 135 960, 964 1376 7 47 1093 - 1095, 1098, 1100 -1103
1234 149 168 960, 964 1376 49 105 1093 - 1095, 1098, 1100 -1103
1234 170 350 960, 964 1376 107 169 1093 - 1095, 1098, 1100 -1103
1235 175 260 960, 963, 964 1371 124 148 1093 - 1095, 1098, 1100 -1103
1235 274 293 960, 963, 964 1372 58 82 1093 - 1095, 1098, 1100 -1103
1235 295 351 960, 963, 964 1373 114 138 1093 - 1095, 1098, 1100 -1103
1236 190 275 960, 963, 964 1374 121 145 1093 - 1095, 1098, 1100 -1103
1236 289 308 960, 963, 964 1375 94 118 1093 - 1095, 1098, 1100 -1103
1236 310 351 960, 963, 964 1376 49 73 1093 - 1095, 1098, 1100 -1103
1237 269 350 960, 963, 964 1371 182 276 1093 - 1095, 1098, 1100 -1103
1238 51 136 960, 964 1371 278 333 1093 - 1095, 1098, 1100 -1103
1238 150 169 960, 964 1372 116 210 1093 - 1095, 1098, 1100 -1103
1238 171 350 960, 964 1372 212 267 1093 - 1095, 1098, 1100 -1103
1239 50 135 960, 964 1372 269 309 1093 - 1095, 1098, 1100 -1103
1239 149 168 960, 964 1372 311 352 1093 - 1095, 1098, 1100 -1103
1239 170 351 960, 964 1373 172 266 1093 - 1095, 1098, 1100 -1103
1240 103 188 960, 963, 964 1373 268 323 1093 - 1095, 1098, 1100 -1103
1240 202 221 960, 963, 964 1373 325 351 1093 - 1095, 1098, 1100 -1103
1240 223 351 960, 963, 964 1374 179 273 1093 - 1095, 1098, 1100 -1103
1241 269 351 960, 963, 964 1374 275 330 1093 - 1095, 1098, 1100 -1103
1242 87 172 960, 963, 964 1374 332 351 1093 - 1095, 1098, 1100 -1103
1242 186 205 960, 963, 964 1375 152 246 1093 - 1095, 1098, 1100 -1103
1242 207 351 960, 963, 964 1375 248 303 1093 - 1095, 1098, 1100 -1103
1235 2 23 960, 963, 964 1375 305 345 1093 - 1095, 1098, 1100 -1103
1235 25 114 960, 963, 964 1376 107 201 1093 - 1095, 1098, 1100 -1103
1236 2 38 960, 963, 964 1376 203 258 1093 - 1095, 1098, 1100 -1103
1236 40 129 960, 963, 964 1376 260 300 1093 - 1095, 1098, 1100 -1103
1237 2 39 960, 963, 964 1376 302 342 1093 - 1095, 1098, 1100 -1103
1237 41 117 960, 963, 964 1371 278 303 1093 - 1095, 1098, 1100 -1103
1237 119 208 960, 963, 964 1372 212 237 1093 - 1095, 1098, 1100 -1103
1240 2 42 960, 963, 964 1373 268 293 1093 - 1095, 1098, 1100 -1103
1241 2 39 960, 963, 964 1374 275 300 1093 - 1095, 1098, 1100 -1103
1241 41 117 960, 963, 964 1375 248 273 1093 - 1095, 1098, 1100 -1103
1241 119 208 960, 963, 964 1376 203 228 1093 - 1095, 1098, 1100 -1103
1242 2 26 960, 963, 964 1371 124 155 1093 - 1095, 1098, 1100 -1103
1234 38 135 960, 964 1372 58 89 1093 - 1095, 1098, 1100 -1103
1234 170 211 960, 964 1373 114 145 1093 - 1095, 1098, 1100 -1103
1235 25 136 960, 963, 964 1374 121 152 1093 - 1095, 1098, 1100 -1103
1235 163 260 960, 963, 964 1375 94 125 1093 - 1095, 1098, 1100 -1103
1235 295 336 960, 963, 964 1376 49 80 1093 - 1095, 1098, 1100 -1103
1236 1 38 960, 963, 964 1371 182 241 1093 - 1095, 1098, 1100 -1103
1236 40 151 960, 963, 964 1372 116 175 1093 - 1095, 1098, 1100 -1103
1236 178 275 960, 963, 964 1373 172 231 1093 - 1095, 1098, 1100 -1103
1237 81 117 960, 963, 964 1374 179 238 1093 - 1095, 1098, 1100 -1103
1237 119 230 960, 963, 964 1375 152 211 1093 - 1095, 1098, 1100 -1103
1237 257 350 960, 963, 964 1376 107 166 1093 - 1095, 1098, 1100 -1103
1238 39 136 960, 964 1371 278 325 1093 - 1095, 1098, 1100 -1103
1238 171 212 960, 964 1372 212 259 1093 - 1095, 1098, 1100 -1103
1239 38 135 960, 964 1373 268 315 1093 - 1095, 1098, 1100 -1103
1239 170 211 960, 964 1374 275 322 1093 - 1095, 1098, 1100 -1103
1240 2 64 960, 963, 964 1375 248 295 1093 - 1095, 1098, 1100 -1103
1240 91 188 960, 963, 964 1376 203 250 1093 - 1095, 1098, 1100 -1103
1240 223 264 960, 963, 964 1309 1 69 1114, 1115
1241 81 117 960, 963, 964 1309 71 96 1114, 1115
1241 119 230 960, 963, 964 1309 98 144 1114, 1115
1241 257 351 960, 963, 964 1309 152 195 1114, 1115 1242 2 48 960, 963, 964 1309 197 237 1114,1115
1242 75 172 960, 963, 964 1309 254 352 1114,1115
1242 207 248 960, 963, 964 1310 25 92 1114,1115
1243 118 158 965, 966, 968, 969 1310 94 119 1114,1115
1243 283 304 965, 966, 968, 969 1310 121 167 1114,1115
1243 310 348 965, 966, 968, 969 1310 175 218 1114,1115
1244 165 205 965, 966, 968, 969 1310 220 260 1114,1115
1244 330 351 965, 966, 968, 969 1310 277 351 1114,1115
1246 205 245 965, 966, 967, 968, 969 1311 7 74 1114,1115
1247 206 246 965, 966, 967, 968, 969 1311 76 101 1114,1115
1248 163 203 965, 966, 968, 969 1311 103 149 1114,1115
1248 328 349 965, 966, 968, 969 1311 157 200 1114,1115
1245 1 20 967 1311 202 242 1114,1115
1245 22 42 967 1311 259 351 1114,1115
1245 104 135 967 1312 9 76 1114,1115
1245 137 155 967 1312 78 103 1114,1115
1245 187 209 967 1312 105 151 1114,1115
1246 36 58 965, 966, 967, 968, 969 1312 159 202 1114,1115
1247 37 59 965, 966, 967, 968, 969 1312 204 244 1114,1115
1244 330 352 965, 966, 968, 969 1312 261 352 1114,1115
1249 2 20 970, 973, 974, 978, 979 1309 2 69 1114,1115
1250 5 84 969 - 971, 973 - 979 1309 254 327 1114,1115
1250 86 270 969 - 971, 973 - 979 1310 22 92 1114,1115
1250 278 306 969 - 971, 973 - 979 1310 277 350 1114,1115
1250 326 351 969 - 971, 973 - 979 1311 4 74 1114,1115
1251 2 52 969 - 971, 973 - 979 1311 259 332 1114,1115
1251 54 238 969 - 971, 973 - 979 1312 6 76 1114,1115
1251 246 274 969 - 971, 973 - 979 1312 261 334 1114,1115
1251 294 322 969 - 971, 973 - 979 1315 56 87 1117,1119-1125
1253 115 194 969 - 971, 973 - 979 1315 91 114 1117,1119-1125
1253 196 351 969 - 971, 973 - 979 1315 116 144 1117,1119-1125
1254 3 31 970, 971, 973, 974, 977 - 979 1315 146 171 1117,1119-1125
1254 51 79 970, 971, 973, 974, 977 - 979 1315 185 225 1117,1119-1125
970,971,973-978, 1626,
1255 282 352 1629 1315 275 294 1117,1119-1125
1256 1 53 970,971,973-978 1315 317 342 1117,1119-1125
1256 55 239 970,971,973-978 1316 22 45 1117,1119-1125
1256 247 275 970,971,973-978 1316 47 75 1117,1119-1125
1256 295 323 970,971,973-978 1316 77 102 1117,1119-1125
1257 6 85 970,971,973-978 1316 116 156 1117,1119-1125
1257 87 271 970,971,973-978 1316 206 225 1117,1119-1125
1257 279 307 970,971,973-978 1316 248 273 1117,1119-1125
1257 327 351 970,971,973-978 1316 275 317 1117,1119-1125
1250 1 84 969 - 971, 973 - 979 1318 144 175 1117,1119-1125
1250 326 352 969 - 971, 973 - 979 1318 179 202 1117,1119-1125
1251 294 319 969 - 971, 973 - 979 1318 204 232 1117,1119-1125
1253 112 194 969 - 971, 973 - 979 1318 234 259 1117,1119-1125
1254 51 76 970, 971, 973, 974, 977 - 979 1318 273 313 1117,1119-1125
970, 971,973-978, 1626,
1255 279 352 1629 1319 2 20 1117,1119-1125
1256 295 320 970,971,973-978 1319 24 47 1117,1119-1125
1257 3 85 970,971,973-978 1319 49 77 1117,1119-1125
1252 31 89 972 1319 79 104 1117,1119-1125
1252 91 236 972 1319 118 158 1117,1119-1125
1252 238 266 972 1319 208 227 1117,1119-1125
1252 277 337 972 1319 250 275 1117,1119-1125
1249 2 23 970, 973, 974, 978, 979 1319 277 319 1117,1119-1125
1249 25 44 970, 973, 974, 978, 979 1313 17 81 1118,1125
1250 34 84 969 - 971, 973 - 979 1313 92 117 1118,1125
1251 1 52 969 - 971, 973 - 979 1313 137 204 1118,1125 1251 294 325 969 - 971, 973 - 979 1313 215 240 1118,1125
1251 327 346 969 - 971, 973 - 979 1313 242 291 1118,1125
1253 144 194 969 - 971, 973 - 979 1313 293 329 1118,1125
1254 51 82 970, 971, 973, 974, 977 - 979 1314 22 89 1118-1125
1254 84 103 970, 971, 973, 974, 977 - 979 1314 100 125 1118-1125
970, 971,973-978, 1626,
1255 311 352 1629 1314 127 176 1118-1125
1256 3 53 970,971,973-978 1314 178 214 1118-1125
1256 295 326 970,971,973-978 1317 25 92 1118, 1120 - 1122, 1124, 1125
1256 328 347 970,971,973-978 1317 103 128 1118, 1120 - 1122, 1124, 1125
1257 35 85 970,971,973-978 1317 130 179 1118, 1120 - 1122, 1124, 1125
1250 47 84 969 - 971, 973 - 979 1317 181 217 1118, 1120 - 1122, 1124, 1125
1251 15 52 969 - 971, 973 - 979 1314 332 351 1118-1125
1253 157 194 969 - 971, 973 - 979 1315 27 51 1117,1119-1125
970, 971,973-978, 1626,
1255 324 352 1629 1316 275 306 1117,1119-1125
1256 16 53 970,971,973-978 1318 115 139 1117,1119-1125
1257 48 85 970,971,973-978 1319 277 308 1117,1119-1125
1250 2 84 969 - 971, 973 - 979 1314 330 351 1118-1125
1250 86 241 969 - 971, 973 - 979 1315 25 51 1117,1119-1125
1251 54 209 969 - 971, 973 - 979 1316 275 303 1117,1119-1125
1253 1 68 969 - 971, 973 - 979 1317 333 351 1118, 1120 - 1122, 1124, 1125
1253 91 194 969 - 971, 973 - 979 1318 113 139 1117,1119-1125
1253 196 352 969 - 971, 973 - 979 1319 277 305 1117,1119-1125
970, 971,973-978, 1626,
1255 169 235 1629 1314 262 293 1118-1125
970, 971,973-978, 1626,
1255 258 352 1629 1314 295 314 1118-1125
1256 55 210 970,971,973-978 1314 316 351 1118-1125
1257 1 85 970,971,973-978 1315 11 51 1117,1119-1125
1257 87 242 970,971,973-978 1317 265 296 1118, 1120 - 1122, 1124, 1125
1250 86 110 969 - 971, 973 - 979 1317 298 317 1118, 1120 - 1122, 1124, 1125
1251 54 78 969 - 971, 973 - 979 1317 319 351 1118, 1120 - 1122, 1124, 1125
1253 2 68 969 - 971, 973 - 979 1318 45 76 1117,1119-1125
1253 196 220 969 - 971, 973 - 979 1318 78 97 1117,1119-1125
970, 971,973-978, 1626,
1255 39 130 1629 1318 99 139 1117,1119-1125
970, 971,973-978, 1626,
1255 132 235 1629 1316 275 324 1117,1119-1125
1256 55 79 970,971,973-978 1319 277 326 1117,1119-1125
1257 87 111 970,971,973-978 1299 50 96 1126-1131,1651-1653
1250 326 350 969 - 971, 973 - 979 1300 2 43 1126-1131,1651-1653
1251 294 318 969 - 971, 973 - 979 1300 66 112 1126-1131,1651-1653
1253 110 194 969 - 971, 973 - 979 1300 123 154 1126-1131,1651-1653
1254 51 75 970, 971, 973, 974, 977 - 979 1300 183 211 1126-1131,1651-1653
970, 971,973-978, 1626,
1255 277 352 1629 1300 213 232 1126-1131,1651-1653
1256 295 319 970,971,973-978 1300 264 310 1126-1131,1651-1653
1257 327 352 970,971,973-978 1301 1 35 1126-1131
1250 32 84 969 - 971, 973 - 979 1301 37 84 1126-1131
1253 142 194 969 - 971, 973 - 979 1301 107 153 1126-1131
970, 971,973-978, 1626,
1255 309 352 1629 1301 164 195 1126-1131
1257 33 85 970,971,973-978 1301 224 252 1126-1131
1249 46 68 970, 973, 974, 978, 979 1301 254 273 1126-1131
1250 61 84 969 - 971, 973 - 979 1301 305 351 1126-1131
1251 29 52 969 - 971, 973 - 979 1302 26 54 1126-1131,1651-1653
1253 171 194 969 - 971, 973 - 979 1302 56 75 1126-1131,1651-1653
1254 105 127 970, 971, 973, 974, 977 - 979 1302 107 153 1126-1131,1651-1653
1256 30 53 970,971,973-978 1303 11 39 1126-1131,1651-1653 1257 62 85 970,971,973-978 1303 41 60 1126-1131,1651-1653
1259 192 226 999 - 1004 1303 92 138 1126-1131,1651-1653
1259 249 298 999 - 1004 1299 50 114 1126-1131,1651-1653
1259 321 352 999 - 1004 1299 131 162 1126-1131,1651-1653
1260 190 224 999 - 1004 1299 215 236 1126-1131,1651-1653
1260 247 296 999 - 1004 1299 248 270 1126-1131,1651-1653
1260 319 351 999 - 1004 1299 272 309 1126-1131,1651-1653
1261 99 133 999 - 1004 1300 264 328 1126-1131,1651-1653
1261 156 205 999 - 1004 1302 107 171 1126-1131,1651-1653
1261 228 289 999 - 1004 1302 188 219 1126-1131,1651-1653
1261 300 334 999 - 1004 1302 272 293 1126-1131,1651-1653
1262 82 116 999 - 1004 1302 305 327 1126-1131,1651-1653
1262 139 188 999 - 1004 1302 329 351 1126-1131,1651-1653
1262 211 272 999 - 1004 1303 92 156 1126-1131,1651-1653
1262 283 317 999 - 1004 1303 173 204 1126-1131,1651-1653
1262 319 350 999 - 1004 1303 257 278 1126-1131,1651-1653
1263 324 351 999 - 1004 1303 290 312 1126-1131,1651-1653
1264 191 225 999 - 1004 1303 314 351 1126-1131,1651-1653
1264 248 297 999 - 1004 1299 272 352 1126-1131,1651-1653
1264 320 351 999 - 1004 1299 272 344 1126-1131,1651-1653
1265 98 132 999 - 1004 1299 272 318 1126-1131,1651-1653
1265 155 204 999 - 1004 1299 272 310 1126-1131,1651-1653
1265 227 288 999 - 1004 1304 2 25 1132, 1134, 1135, 1136
1265 299 333 999 - 1004 1304 36 169 1132, 1134, 1135, 1136
1259 95 145 999 - 1004 1304 186 214 1132, 1134, 1135, 1136
1259 165 226 999 - 1004 1304 231 250 1132, 1134, 1135, 1136
1260 93 143 999 - 1004 1304 264 307 1132, 1134, 1135, 1136
1260 163 224 999 - 1004 1306 2 70 1132, 1134, 1135, 1136
1261 1 52 999 - 1004 1306 87 115 1132, 1134, 1135, 1136
1261 72 133 999 - 1004 1306 132 151 1132, 1134, 1135, 1136
1262 1 35 999 - 1004 1306 165 208 1132, 1134, 1135, 1136
1262 55 116 999 - 1004 1307 1 27 1132, 1134, 1135, 1136
1263 227 277 999 - 1004 1307 29 69 1132, 1134, 1135, 1136
1263 297 351 999 - 1004 1307 80 213 1132, 1134, 1135, 1136
1264 94 144 999 - 1004 1307 230 258 1132, 1134, 1135, 1136
1264 164 225 999 - 1004 1307 275 294 1132, 1134, 1135, 1136
1265 2 51 999 - 1004 1307 308 351 1132, 1134, 1135, 1136
1265 71 132 999 - 1004 1308 2 33 1132, 1134, 1135, 1136
1259 15 40 999 - 1004 1308 44 177 1132, 1134, 1135, 1136
1259 42 64 999 - 1004 1308 194 222 1132, 1134, 1135, 1136
1259 66 88 999 - 1004 1308 239 258 1132, 1134, 1135, 1136
1259 90 145 999 - 1004 1308 272 315 1132, 1134, 1135, 1136
1259 165 219 999 - 1004 1347 2 28 1132, 1134, 1135, 1136
1260 13 38 999 - 1004 1347 39 172 1132, 1134, 1135, 1136
1260 40 62 999 - 1004 1347 189 217 1132, 1134, 1135, 1136
1260 64 86 999 - 1004 1347 234 253 1132, 1134, 1135, 1136
1260 88 143 999 - 1004 1347 267 310 1132, 1134, 1135, 1136
1133, 1137, 1138, 1580, 1581,
1260 163 217 999 - 1004 1305 1 133 1583
1133, 1137, 1138, 1580, 1581,
1261 2 52 999 - 1004 1305 135 154 1583
1133, 1137, 1138, 1580, 1581,
1261 72 126 999 - 1004 1305 156 178 1583
1133, 1137, 1138, 1580, 1581,
1262 55 109 999 - 1004 1305 195 280 1583
1133, 1137, 1138, 1580, 1581,
1263 1 60 999 - 1004 1305 330 351 1583
1133, 1137, 1138, 1580, 1581,
1263 105 127 999 - 1004 1344 13 35 1583
1263 147 172 999 - 1004 1344 52 137 1133, 1137, 1138, 1580, 1581, 1583
1133, 1137, 1138, 1580, 1581,
1263 174 196 999 - 1004 1344 187 208 1583
1133, 1137, 1138, 1580, 1581,
1263 198 220 999 - 1004 1345 50 181 1583
1133, 1137, 1138, 1580, 1581,
1263 222 277 999 - 1004 1345 183 202 1583
1133, 1137, 1138, 1580, 1581,
1263 297 352 999 - 1004 1345 204 226 1583
1133, 1137, 1138, 1580, 1581,
1264 14 39 999 - 1004 1345 243 328 1583
1264 41 63 999 - 1004 1346 2 81 1133, 1137, 1138
1264 65 87 999 - 1004 1346 131 152 1133, 1137, 1138
1264 89 144 999 - 1004 1304 1 25 1132, 1134, 1135, 1136
1264 164 218 999 - 1004 1304 264 334 1132, 1134, 1135, 1136
1265 71 125 999 - 1004 1306 165 235 1132, 1134, 1135, 1136
1259 190 226 999 - 1004 1307 46 69 1132, 1134, 1135, 1136
1260 188 224 999 - 1004 1308 10 33 1132, 1134, 1135, 1136
1261 97 133 999 - 1004 1308 272 342 1132, 1134, 1135, 1136
1262 80 116 999 - 1004 1347 5 28 1132, 1134, 1135, 1136
1263 322 351 999 - 1004 1347 267 337 1132, 1134, 1135, 1136
1264 189 225 999 - 1004 1304 101 169 1132, 1134, 1135, 1136
1265 96 132 999 - 1004 1306 1 70 1132, 1134, 1135, 1136
1259 96 145 999 - 1004 1306 237 310 1132, 1134, 1135, 1136
1260 94 143 999 - 1004 1306 312 352 1132, 1134, 1135, 1136
1261 3 52 999 - 1004 1307 145 213 1132, 1134, 1135, 1136
1263 228 277 999 - 1004 1308 109 177 1132, 1134, 1135, 1136
1264 95 144 999 - 1004 1347 104 172 1132, 1134, 1135, 1136
1265 1 51 999 - 1004 1307 38 69 1132, 1134, 1135, 1136
1259 165 218 999 - 1004 1308 1 33 1132, 1134, 1135, 1136
1133, 1137, 1138, 1580, 1581,
1260 163 216 999 - 1004 1344 187 239 1583
1133, 1137, 1138, 1580, 1581,
1261 72 125 999 - 1004 1344 241 272 1583
1133, 1137, 1138, 1580, 1581,
1262 55 108 999 - 1004 1344 286 308 1583
1133, 1137, 1138, 1580, 1581,
1263 2 60 999 - 1004 1344 310 329 1583
1263 297 350 999 - 1004 1346 131 183 1133, 1137, 1138
1264 164 217 999 - 1004 1346 185 216 1133, 1137, 1138
1265 71 124 999 - 1004 1346 230 252 1133, 1137, 1138
1266 2 39 1016 - 1023 1346 254 273 1133, 1137, 1138
1133, 1137, 1138, 1580, 1581,
1266 41 87 1016 - 1023 1305 227 280 1583
1133, 1137, 1138, 1580, 1581,
1266 89 129 1016 - 1023 1344 84 137 1583
1133, 1137, 1138, 1580, 1581,
1267 2 74 1016 - 1023 1345 275 328 1583
1267 76 122 1016 - 1023 1346 28 81 1133, 1137, 1138
1267 124 164 1016 - 1023 1346 290 351 1133, 1137, 1138
1268 2 53 1016 - 1023 1348 44 66 1139, 1140, 1141, 1142
1268 55 101 1016 - 1023 1348 104 129 1139, 1140, 1141, 1142
1268 103 143 1016 - 1023 1348 131 156 1139, 1140, 1141, 1142
1269 2 72 1016 - 1023 1348 158 195 1139, 1140, 1141, 1142
1269 74 120 1016 - 1023 1348 167 189 1139, 1140, 1141, 1142
1269 122 162 1016 - 1023 1348 197 237 1139, 1140, 1141, 1142
1270 2 107 1016 - 1023 1348 215 234 1139, 1140, 1141, 1142
1270 109 155 1016 - 1023 1348 4 49 1139, 1140, 1141, 1142
1270 157 197 1016 - 1023 1348 4 74 1139, 1140, 1141, 1142
1271 2 62 1016 - 1023 1348 4 85 1139, 1140, 1141, 1142 1271 64 110 1016 - 1023 1349 19 90 1143
1271 112 152 1016 - 1023 1349 146 207 1143
1266 131 177 1016 - 1023 1349 209 256 1143
1266 179 207 1016 - 1023 1350 32 51 1144-1151,1703
1266 209 243 1016 - 1023 1350 71 90 1144-1151,1703
1267 166 212 1016 - 1023 1351 118 137 1144-1151
1267 214 242 1016 - 1023 1351 157 176 1144-1151
1267 244 278 1016 - 1023 1353 81 100 1144-1151
1268 145 191 1016 - 1023 1353 120 139 1144-1151
1268 193 221 1016 - 1023 1354 1 42 1144-1151
1268 223 257 1016 - 1023 1354 218 237 1144-1151
1269 164 210 1016 - 1023 1354 257 276 1144-1151
1269 212 240 1016 - 1023 1356 43 62 1144-1151
1269 242 276 1016 - 1023 1356 82 101 1144-1151
1270 199 245 1016 - 1023 1350 176 201 1144-1151,1703
1270 247 275 1016 - 1023 1350 209 231 1144-1151,1703
1270 277 311 1016 - 1023 1350 236 267 1144-1151,1703
1271 154 200 1016 - 1023 1351 262 287 1144-1151
1271 202 230 1016 - 1023 1351 295 317 1144-1151
1271 232 266 1016 - 1023 1351 322 351 1144-1151
1266 209 294 1016 - 1023 1353 225 250 1144-1151
1266 296 316 1016 - 1023 1353 258 280 1144-1151
1267 1 74 1016 - 1023 1353 285 316 1144-1151
1267 244 329 1016 - 1023 1356 187 212 1144-1151
1267 331 351 1016 - 1023 1356 220 242 1144-1151
1268 223 308 1016 - 1023 1356 247 278 1144-1151
1268 310 330 1016 - 1023 1350 236 265 1144-1151,1703
1269 242 327 1016 - 1023 1351 322 352 1144-1151
1269 329 349 1016 - 1023 1353 285 314 1144-1151
1270 35 107 1016 - 1023 1356 247 276 1144-1151
1270 277 351 1016 - 1023 1350 287 306 1144-1151,1703
1271 232 317 1016 - 1023 1350 323 348 1144-1151,1703
1271 319 339 1016 - 1023 1352 2 21 1147, 1150, 1151, 1703
1266 296 318 1016 - 1023 1352 38 63 1147, 1150, 1151, 1703
1267 4 74 1016 - 1023 1355 5 24 1147, 1150, 1151, 1703
1268 310 332 1016 - 1023 1355 41 66 1147, 1150, 1151, 1703
1269 1 72 1016 - 1023 1356 298 317 1144-1151
1079-1082,1152-1154,
1269 329 351 1016 - 1023 1358 322 351 1455, 1457, 1458
1079-1082,1152-1154,
1270 37 107 1016 - 1023 1364 262 291 1455, 1457, 1458
1079-1082,1152-1154,
1271 319 341 1016 - 1023 1366 229 258 1455, 1457, 1458
1266 131 157 1016 - 1023 1367 154 183 1079-1082,1152-1154
1079-1082,1152-1154,
1267 166 192 1016 - 1023 1361 2 41 1455, 1457, 1458
1268 145 171 1016 - 1023 1210 1 35 1431
1269 164 190 1016 - 1023 1211 30 63 1431
1270 199 225 1016 - 1023 1213 15 48 1431
1271 154 180 1016 - 1023 1217 19 52 1431
1079-1082,1152-1154,
1295 113 168 1026, 1027, 1024 1357 162 200 1455, 1457, 1458
1079-1082,1152-1154,
1295 170 201 1026, 1027, 1024 1358 124 162 1455, 1457, 1458
1079-1082,1152-1154,
1295 203 233 1026, 1027, 1024 1359 130 168 1455, 1457, 1458
1079-1082,1152-1154,
1295 236 258 1026, 1027, 1024 1360 137 175 1455, 1457, 1458
1079-1082,1152-1154,
1297 128 183 1026, 1027, 1024 1361 163 201 1455, 1457, 1458 1079 - 1082, 1152 - 1154,
1297 185 216 1026, 1027, 1024 1362 130 168 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1297 218 248 1026, 1027, 1024 1363 132 170 1455, 1457, 1458
1025, 1028, 1029, 1515 - 1079 - 1082, 1152 - 1154,
1296 1 44 1518, 1520 1364 64 102 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1298 7 57 1025, 1028, 1029 1365 134 172 1455, 1457, 1458
1079 - 1082, 1152 - 1154,
1298 59 96 1025, 1028, 1029 1366 31 69 1455, 1457, 1458
1298 110 132 1025, 1028, 1029 1054 198 244 1492, 1496, 1497, 1498
1298 143 210 1025, 1028, 1029 1054 285 319 1492, 1496, 1497, 1498
1295 17 87 1026, 1027, 1024 1056 191 237 1492, 1496, 1497, 1498
1297 32 102 1026, 1027, 1024 1056 278 312 1492, 1496, 1497, 1498
1294 127 153 1027 1057 14 60 1492, 1496, 1497, 1498
1295 17 46 1026, 1027, 1024 1057 101 135 1492, 1496, 1497, 1498
1297 32 61 1026, 1027, 1024 1057 233 264 1492, 1496, 1497, 1498
1298 5 57 1025, 1028, 1029 1060 269 315 1492, 1496, 1497, 1498
1054 - 1057, 1060, 1492, 1496
1320 249 268 1033, 1035, 1076 1390 10 50 - 1498
1054 - 1057, 1060, 1492, 1496
1324 2 23 1036, 1037, 1038 1390 142 173 - 1498
1054 - 1057, 1060, 1492, 1496
1326 14 38 1036, 1037, 1038 1391 73 104 - 1498
1326 95 117 1036, 1037, 1038 1055 85 131 1493, 1494, 1495, 1499, 1500
1326 152 174 1036, 1037, 1038 1058 117 163 1493, 1494, 1495, 1499, 1500
1326 227 248 1036, 1037, 1038 1059 118 164 1493, 1494, 1495, 1499, 1500
1327 44 65 1036, 1037, 1038 1061 109 155 1493, 1494, 1495, 1499, 1500
1321 65 86 1037 1062 222 268 1493, 1494, 1495, 1499, 1500
1321 88 132 1037 1063 262 308 1493, 1494, 1495, 1499, 1500
1322 2 34 1037 1054 15 64 1492, 1496, 1497, 1498
1322 36 67 1037 1054 165 196 1492, 1496, 1497, 1498
1322 135 156 1037 1056 8 57 1492, 1496, 1497, 1498
1322 158 202 1037 1056 158 189 1492, 1496, 1497, 1498
1323 1 35 1037 1060 86 135 1492, 1496, 1497, 1498
1323 37 68 1037 1060 236 267 1492, 1496, 1497, 1498
1323 136 157 1037 1386 199 248 1054 - 1057, 1060, 1496, 1498
1323 159 203 1037 1388 212 261 1054 - 1057, 1060, 1496, 1498
1054 - 1057, 1060, 1492, 1496
1324 2 51 1036, 1037, 1038 1391 286 335 - 1498
1055, 1058, 1059, 1061 - 1063,
1324 56 93 1036, 1037, 1038 1384 311 351 1500
1055, 1058, 1059, 1061 - 1063,
1324 95 126 1036, 1037, 1038 1385 312 351 1500
1055, 1058, 1059, 1061 - 1063,
1324 194 215 1036, 1037, 1038 1387 83 123 1500, 1057
1055, 1058, 1059, 1061 - 1063,
1324 217 261 1036, 1037, 1038 1389 162 202 1500
1025, 1028, 1029, 1515, 1516,
1325 2 31 1037 1296 69 101 1517, 1518, 1520
1325 33 64 1037 909 305 340 1531
1325 132 153 1037 960 70 101 1543, 1551
1325 155 199 1037 964 259 290 1543, 1551
1133, 1137, 1138, 1580, 1581,
1326 227 276 1036, 1037, 1038 1305 180 226 1583
1326 281 318 1036, 1037, 1038 1133 195 226 1580, 1581
1326 320 350 1036, 1037, 1038 1137 52 83 1580, 1581
1133, 1137, 1138, 1580, 1581,
1327 44 93 1036, 1037, 1038 1344 37 83 1583
1327 98 135 1036, 1037, 1038 1345 228 274 1133, 1137, 1138, 1580, 1581, 1583
1133, 1137, 1138, 1580, 1581,
1327 137 168 1036, 1037, 1038 1305 180 217 1583
1133, 1137, 1138, 1580, 1581,
1327 236 257 1036, 1037, 1038 1344 37 74 1583
1133, 1137, 1138, 1580, 1581,
1327 259 303 1036, 1037, 1038 1345 228 265 1583
1324 2 24 1036, 1037, 1038 1039 176 210 1590, 1594, 1597, 1598
1326 227 249 1036, 1037, 1038 1040 142 176 1590, 1594, 1597, 1598
1327 44 66 1036, 1037, 1038 1041 248 282 1590, 1594, 1597, 1598
1039 - 1043, 1045, 1594,
1328 3 59 1597, 1598 1042 141 175 1590, 1594, 1597, 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1328 70 146 1597, 1598 1328 175 209 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1328 148 215 1597, 1598 1331 2 54 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1328 232 263 1597, 1598 1331 200 234 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1328 328 350 1597, 1598 1332 142 176 1598
1039 - 1043, 1045, 1594, 1597,
1329 22 53 1039 - 1043 1045 1333 176 210 1598
1039 - 1043, 1045, 1594, 1597,
1329 118 140 1039 - 1043 1045 1334 16 68 1598
1039 - 1043, 1045, 1594, 1597,
1330 31 62 1039 - 1043 1045 1334 214 248 1598
1039 - 1043, 1045, 1594, 1597,
1330 127 149 1039 - 1043 1045 1335 76 110 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1331 28 84 1597, 1598 1336 8 60 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1331 95 171 1597, 1598 1336 206 240 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1331 173 240 1597, 1598 1338 14 66 1598
1039 - 1043, 1045, 1594, 1039 - 1043, 1045, 1594, 1597,
1331 257 288 1597, 1598 1338 212 246 1598
1039 - 1043, 1045, 1594, 970, 971, 973 - 978, 1626,
1332 2 26 1597, 1598 1255 165 199 1629
1039 - 1043, 1045, 1594,
1332 37 113 1597, 1598 976 129 163 1626, 1629
1039 - 1043, 1045, 1594,
1332 115 182 1597, 1598 1299 98 129 1126 - 1131, 1651 - 1653
1039 - 1043, 1045, 1594,
1332 199 230 1597, 1598 1300 312 343 1126 - 1131, 1651 - 1653
1039 - 1043, 1045, 1594,
1332 295 317 1597, 1598 1302 155 186 1126 - 1131, 1651 - 1653
1039 - 1043, 1045, 1594,
1333 4 60 1597, 1598 1303 140 171 1126 - 1131, 1651 - 1653
1039 - 1043, 1045, 1594,
1333 71 147 1597, 1598 1350 314 345 1144 - 1151, 1703
1039 - 1043, 1045, 1594,
1333 149 216 1597, 1598 1352 29 60 1147, 1150, 1151, 1703
1039 - 1043, 1045, 1594,
1333 233 264 1597, 1598 1355 32 63 1147, 1150, 1151, 1703 Example 8
[00131] This example discloses embodiments related to polynucleotide molecules having a nucleotide sequence containing specific modifications such as nucleotide substitutions. Embodiments of such modifications include modified polynucleotides that provide improved sequence discrimination between the intended target gene of the insect pest of interest, and genetic sequences of other, non- target species.
[00132] Double-stranded RNAs identified in Table 1 were screened for sequence matches to a sequence of at least 19 contiguous nucleotides identified in a non-target gene or a non-target organism (NTO, e. g., Apis mellifera, Bombus impatiens and B. terrestris; Bombyx mori; Bos taurus; Canis lupus familiaris; Coleomegilla maculata; Danio rerio; Danaus plexippus; Daphnia pulex; Equus caballus; Gallus gallus; Homo sapiens; Megachile rotundata; Mus musculus; Sus scrofa; Brassica napus;
Brassica oleracea; Brassica rapa; Glycine max; Gossypium raimondii; Solanum tuberosum; Solanum lycopersicum; and Zea mays). Nucleotide changes are made to eliminate contiguous sequence matches over 19 nucleotides to a non-target gene or non-target organism. Examples of such modified polynucleotide sequences are provided by SEQ ID NO:1725, which corresponds to SEQ ID NO:870 (which targets the target gene having SEQ ID NO:ll), SEQ ID NO:1726, which corresponds to SEQ ID NO: 1224 (which targets the target gene having SEQ ID NO:365), SEQ ID NO: 1727, which corresponds to SEQ ID NO:875 (which targets the target gene having SEQ ID NO:16), SEQ ID NO:1728, which corresponds to SEQ ID NO:1193 (which targets the target gene having SEQ ID NO:334), SEQ ID NO: 1729, which corresponds to SEQ ID NO:871 (which targets the target gene having SEQ ID NO:12), and SEQ ID NO:1730, which corresponds to SEQ ID NO:1187 (which targets the target gene having SEQ ID NO:328).
[00133] These modified polynucleotide sequences SEQ ID NOs:1725 - 1730 are useful for designing polynucleotides for providing improved discrimination between the intended target species and non-target organisms. For example, embodiments of insecticidal compositions useful for controlling or preventing flea beetle infestations comprise an effective amount of a dsRNA comprising one RNA strand having a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730, or a fragment thereof. In embodiments, insecticidal compositions for causing stunting or mortality in flea beetles comprise an effective amount of a dsRNA comprising one RNA strand having at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730. In embodiments, insecticidal compositions for causing stunting or mortality in flea beetles comprise an effective amount of an RNA molecule comprising at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730 wherein the at least one segment is embedded in larger sections of neutral sequence to provide an efficacious insecticidal polynucleotide. In another embodiment, segments of at least 21 contiguous nucleotides from multiple sequences selected from the group consisting of SEQ ID NOs:1725 - 1730 are embedded in larger sections of neutral sequence to provide an efficacious insecticidal
polynucleotide for controlling flea beetles. Methods for selecting efficacious insecticidal polynucleotides are described elsewhere in this application, e. g. , the method including oral delivery of insecticidal polynucleotides to flea beetles, resulting in stunting or mortality in the flea beetles, described in Example 5. These modified polynucleotide sequences SEQ ID NOs:1725 - 1730 are useful in a method of causing mortality or stunting in an insect, comprising providing in the diet of an insect, such as a flea beetle, at least one polynucleotide that comprises at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730, wherein ingestion of the polynucleotide by the insect results in mortality or stunting in the insect.; in embodiments, the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730. Polynucleotides comprising a sequence of SEQ ID NOs:1725 - 1730 are useful in a method for controlling an insect infestation of a plant comprising contacting, with a polynucleotide, an insect that infests a plant, wherein the polynucleotide comprises at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730, whereby the insect infestation is controlled; in embodiments, the polynucleotide comprisesa sequence selected from the group consisting of SEQ ID NOs:1725 - 1730. Polynucleotides as described herein are useful in a method of providing a plant having improved resistance to an insect, comprising expressing in the plant a recombinant DNA construct, wherein the recombinant DNA encodes a polynucleotide that comprises at least one segment of at least 21 contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730, wherein ingestion of the
polynucleotide by the insect results in mortality or stunting in the insect.; in some embodiments, the polynucleotide comprises a sequence selected from the group consisting of SEQ ID NOs:1725 - 1730.
Example 9
[00134] This example discloses additional insecticidal polynucleotide molecules with complementarity to both Phyllotreta cruciferae and Psylloides striolata. A total of two hundred-forty (240) polynucleotide sequences were engineered (SEQ ID NOs 1731-1971). These polynucleotides target gene transcripts for calmodulin, casein kinase la and elongation factori 1 -alpha across both flea beetle species. Fifty-one polynucleotides are tiling polynucleotides that cover the transcript length of these genes. The following Table 5 summarizes these fifty-one tiling polynucleotides and the transcripts targeted.
Table 5 Insecticidal Polynucleotides and target transcripts
Figure imgf000076_0001
1737 TubulinG PHYST-05 JAN16-TRPT0002886 : 5
1738 COPI Delta PHYST-05 JAN16-TRPT0009108:2
1739 COPI Delta PHYST-05JAN16-TRPT0009108:5
1740 COPI Delta PHYST-05JAN16-TRPT0009108:6
1741 COPI Delta PHYST-05 JAN16-TRPT0009108: 11
1742 COPI Delta PHYST-05JAN16-TRPT0009108: 12
1743 sec23 PHYST-05 JAN16-TRPT0011013 : 1
1744 sec23 PHYST-05 JAN16-TRPT0011013 :2
1745 sec23 PHYST-05 JAN16-TRPT0011013 :3
1746 sec23 PHYST-05 JAN16-TRPT0011013 :4
1747 COPI Gamma PHYST-05JAN16-TRPT0018736: 1
1748 COPI Gamma PHYST-05JAN16-TRPT0018736:2
1749 COPI Gamma PHYST-05JAN16-TRPT0018736:3
1750 COPI Gamma PHYST-05JAN16-TRPT0018736:4
1751 COPI Gamma PHYST-05JAN16-TRPT0018736:5
1752 Rpt6 PHYST-05JAN16-TRPT0036466: 1
1753 Rpt6_PHYST-05JAN16-TRPT0036466:2
1754 Rpt6 PHYST-05JAN16-TRPT0036466:3
1755 Rpt6_PHYST-05JAN16-TRPT0036466:4
1756 Rpt6 PHYST-05JAN16-TRPT0036466:5
1757 actin PHYST-05JAN16-TRPT0037252: 1
1758 actin PHYST-05JAN16-TRPT0037252:3
1759 actin PHYST-05JAN16-TRPT0037252:5
1760 vATPaseA PHYST-05 JAN16-TRPT0041323: 1
1761 RpL13 PHYST-05JAN16-TRPT0045203 : 1
1762 RpL13_PHYST-05JAN16-TRPT0045203 :2
1763 RpS14 PHYST-05JAN16-TRPT0045884: 1
1764 RpS14_PHYST-05JAN16-TRPT0045884:2
1765 actin PHYCR-05JAN16-TRPT0000169:2
1766 actin PHYCR-05JAN16-TRPT0000169:3
1767 vATPase-A PHYCR-05JAN16-TRPT0001064: 1
1768 vATPase-A PHYCR-05 JAN16-TRPT0001064 : 3
1769 vATPase-A PHYCR-05 JAN16-TRPT0001064 :4
1770 vATPase-A PHYCR-05 JAN16-TRPT0001064 : 5
1771 vATPase-A PHYCR-05 JAN16-TRPT0001064 : 6
1772 vATPase-A PHYCR-05 JAN16-TRPT0001064 : 7
1773 vATPase-A PHYCR-05 JAN16-TRPT0001064 : 8
1774 tubulinG PHYCR-05JAN16-TRPT0001496: 1
1775 tubulinG PHYCR-05JAN16-TRPT0001496:2
1776 tubulinG PHYCR-05JAN16-TRPT0001496:3
1777 tubulinG PHYCR-05JAN16-TRPT0001496:4
1778 tubulinG PHYCR-05 J AN 16-TRPT0001496:5
1779 tubulinG PHYCR-05JAN16-TRPT0001496:6
1780 RpS14 PHYCR-05JAN16-TRPT0002697: 1
1782 RpS14 PHYCR-05 JAN16-TRPT0002697:2 Example 10
[00135] This example illustrates non-limiting embodiments of methods of testing the efficacy of insecticidal polynucleotides in flea beetles. More specifically this example illustrates a method comprising oral delivery of dsRNAs to flea beetles, resulting in stunting or mortality in the flea beetles.
[00136] P. cruciferae and P. striolata were collected from a canola field where no pesticides had been applied in the previous 3 months. Two dsRNAs (SEQ ID NOs:1972 and 1974) targeting Phyllotreta cruciferae genes (COPI_delta and Rpt6, respectively) and two dsRNAs (SEQ ID Nos: 1973 and 1975) targeting Phyllotreta striolata genes (COPI_delta and Rpt6, respectively) and one negative control dsRNA targeting GFP were tested on groups of 30 P. cruciferae or P '. striolata by using 10 insects in three separate replications. The dsRNAs were resuspended in water and applied to 9 millimeter leaf discs (55 ± 6 milligrams each) at a dose of 50 nanograms dsRNA/milligram leaf tissue, which were fed to groups of 5 flea beetles. Leaf discs with freshly applied dsRNA were replaced every other day, and the number of surviving individuals was recorded over a 12-day period.
[00137] A low non-specific mortality rate was observed in the negative-control insect groups
(4 out of 30 insects dying over 12 days ΐοτ Ρ. cruciferae, or 13% non-specific mortality and 8 out of 30 insects dying over 12 days for P. striolata, or 26% non-specific mortality). Mortality was observed beginning at day 2 and continuing through the 12 day period. Specific mortality was observed for all dsRNA treatments (Table 6). Correcting for non-specific mortality (subtracting non-specific mortality rate of 4 or 8 insects per group for corrected N= 26 or N=22), the percent mortality observed at the end of the 12 day period ranged from 36-38% for the dsRNAs tested. These results demonstrated the efficacy of the dsRNAs in causing mortality in flea beetles when provided in the flea beetles' diet and their cross species effect.
Table 6. Mortality in flea beetles fed an artificial diet containing insecticidal
polynucleotide.
Figure imgf000078_0001
Cumulative mortality (days after treatment, 7V=30)
SEQ Target gene Species dO d2 dS d7 d9 dl2
ID species tested
NO against
1973 striped crucifer 0 0 2 6 6 9
1974 crucifer crucifer 0 1 3 7 8 9
1975 striped crucifer 0 0 0 2 6 8 striped 0 0 1 4 7 8
NA striped 0 0 1 2 6 8
1972 crucifer striped 0 0 3 9 10 10
1973 striped striped 0 0 3 9 9 10
1974 crucifer striped 0 0 3 7 7 8
1975 striped striped 0 1 3 5 8 9
[00138] All of the materials and methods disclosed and claimed herein can be made and used without undue experimentation as instructed by the above disclosure. Although the materials and methods disclosed herein have been described in terms of preferred embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of this invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of this invention as defined by the appended claims.

Claims

What is claimed is:
1. A method of causing mortality or stunting in an insect, comprising providing in the diet of an insect at least one insecticidal polynucleotide comprising at least one silencing element essentially identical or essentially complementary to a fragment of a target gene sequence of said insect, wherein said target gene sequence is selected from the group consisting of SEQ ID NOs:l - 859, and wherein ingestion of said insecticidal polynucleotide by said insect results in mortality or stunting in said insect.
2. The method of claim 1, wherein said silencing element comprises:
(a) at least one segment of 18 or more contiguous nucleotides comprising a sequence of 100% complementarity with a fragment of said target gene of said insect, wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859;
(b) at least one segment of 18 or more contiguous nucleotides of a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1730 and 1771- 1975;
(c) a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and
1722 - 1730 and 1771-1975
3. The method of claim 1, wherein said insecticidal polynucleotide is provided in a
microbial or plant cell that expresses said insecticidal polynucleotide, or in a microbial fermentation product, or is chemically synthesized.
4. The method of claim 1, wherein said at least one insecticidal polynucleotide is provided in a composition comprising said at least one insecticidal polynucleotide, wherein said composition is applied to a surface of said insect or to a surface of a seed or plant in need of protection from infestation by said insect.
5. The method of claim 4, wherein said composition:
(a) comprises a solid, liquid, powder, suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, or seed treatment;
(b) further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator;
(c) further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus {Bacillus laterosporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae; or (d) is ingested by said insect.
6. A method for controlling an insect infestation of a plant comprising contacting, with a dsRNA, an insect that infests a plant, wherein said dsRNA comprises at least one segment of 18 or more contiguous nucleotides of a sequence of about 95% to about 100%) complementarity with a fragment of a target gene of said insect, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859.
7. The method of claim 6, wherein said contacting comprises:
(a) oral delivery to said insect, non-oral delivery to said insect, or a combination of oral and non-oral delivery to said insect;
(b) application of a composition comprising said dsRNA to a surface of said insect or to a surface of said plant infested by said insect;
(c) providing said dsRNA in a composition that further comprises one or more
components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator;
(d) providing said dsRNA in a composition that further comprises at least one
pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus {Bacillus sphaericus), Brevibacillus laterosporus {Bacillus
laterosporus), Chromobacterium species, Chromobacterium subtsugae,
Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae; or
(e) providing said dsRNA in a composition that is ingested by said insect.
8. An insecticidal composition comprising an insecticidally effective amount of a
insecticidal polynucleotide, wherein said insecticidal polynucleotide comprises at least one segment of 18 or more contiguous nucleotides comprising a sequence of about 95% to about 100% complementarity with a fragment of a target gene of an insect that infests a plant, and wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs: l - 859.
9. The insecticidal composition of claim 8, wherein said insecticidal composition:
(a) further comprises one or more components selected from the group consisting of a carrier agent, a surfactant, an organosilicone, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticide, a fungicide, a safener, a fertilizer, a micronutrient, an insect attractant, and an insect growth regulator;
(b) further comprises at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus laterosporus (Bacillus laterosporus), Chromobacterium species, Chromobacterium subtsugae, Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae;
(c) is in a form selected from the group consisting of a solid, liquid, powder,
suspension, emulsion, spray, encapsulation, microbeads, carrier particulates, film, matrix, soil drench, insect diet or insect bait, and seed treatment; or
(d) is provided in a microbial or plant cell that expresses said recombinant RNA, or in a microbial fermentation product.
10. A plant treated with the insecticidal composition of claim 8, or a plant grown from seed treated with the insecticidal composition of claim 8, wherein said plant exhibits improved resistance to said insect.
11. A method of providing a plant having improved resistance to an insect, comprising expressing in said plant a recombinant DNA construct, wherein said recombinant DNA construct comprises DNA encoding an RNA having a sequence essentially identical or essentially complementary to at least 18 or more contiguous nucleotides of said target gene, wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs: l - 859, and wherein ingestion of said RNA by said insect results in mortality or stunting in said insect.
12. The method of claims 1 or 11, or the insecticidal composition of claim 8, wherein said RNA: (a) comprises a strand comprising at least 21 contiguous nucleotides comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1975; or
(b) comprises dsRNA.
13. The method or insecticidal composition of claim 12, wherein:
(a) said dsRNA is blunt-ended, or has an overhang at at least one terminus, or
comprises at least one stem-loop;
(b) said dsRNA is chemically synthesized, produced by expression in a microorganism, produced by expression in a plant cell, or produced by microbial fermentation;
(c) said dsRNA is chemically modified; or
(d) said dsRNA comprises at least one RNA strand comprising a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1730 and 1731-1975.
14. The method of claim 6 or 12, wherein said dsRNA comprises:
(a) a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and
1722 - 1730 and 1731-1975, or the complement thereof;
(b) at least one segment of 18 or more contiguous nucleotides with a sequence of 100% complementarity with a fragment of said target gene of said insect, wherein said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:l - 859; or
(c) at least one segment of 18 or more contiguous nucleotides of an RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1730 and 1731-1975
15. The method of any one of claims 1, 6, or 11, or the insecticidal composition of claim 8, wherein:
(a) said insect is a flea beetle;
(b) said insect is a species of a genus selected from the group consisting of the genera Altica, Anthobiodes, Aphthona, Aphthonaltica, Aphthonoides, Apteopeda,
Argopistes, Argopus, Arrhenocoela, Batophila, Blepharida, Chaetocnema, Clitea, Crepidodera, Derocrepis, Dibolia, Disonycha, Epitrix, Hermipyxis,
Hermaeophaga, Hespera, Hippuriphila, Horaia, Hyphasis, Lipromima, Liprus, Longitarsus, Luperomorpha, Lythraria, Manobia, Mantura, Meishania, Minota, Mniophila, Neicrepidodera, Nonarthra, Novofoudrasia, Ochrosis, Oedionychis, Oglobinia, Omeisphaera, Ophrida, Orestia, Paragopus, Pentamesa, Philopona, Phygasia, Phyllotreta, Podagrica, Podagricomela, Podontia, Pseudodera, Psylliodes, Sangariola, Sinaltica, Sphaeroderma, Systena, Trachyaphthona, Xuthea, and Zipangia;
(c) said insect is a species selected from the group consisting of Altica ambiens (alder flea beetle), Altica canadensis (prairie flea beetle), Altica chalybaea (grape flea beetle), Altica prasina (poplar flea beetle), Altica rosae (rose flea beetle), Altica sylvia (blueberry flea beetle), Altica ulmi (elm flea beetle), Chaetocnema pulicaria (corn flea beele), Chaetocnema conofinis (sweet potato flea beetle), Epitrix cucumeris (potato flea beetle), Systena blanda (palestripped fleabeetle), and Systena frontalis (redheaded flea beetle);
(d) said insect is a species selected from the group consisting of Phyllotreta
armoraciae (horseradish flea beetle), Phyllotreta cruciferae (canola flea beetle), Phyllotreta pusilla (western black flea beetle), Phyllotreta nemorum (striped turnip flea beetle), Phyllotreta atra (turnip flea beetle), Phyllotreta robusta (garden flea beetle), Phyllotreta striolata (striped flea beetle), Phyllotreta undulata, Psylliodes chrysocephala, and Psylliodes punctulata (hop flea beetle);
(e) said insect is an insect pest of a Brassica species;
(f) said insect is a Phyllotreta species and said target gene has a DNA sequence
selected from the group consisting of SEQ ID NOs: l - 551;
(g) said insect is Phyllotreta atra (turnip flea beetle) and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs: l - 296;
(h) said insect is Phyllotreta cruciferae (canola flea beetle) and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:297 - 532;
(i) said insect is Phyllotreta striolata (striped flea beetle) and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:533 - 551;
(i) said insect is a Psylliodes species and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859;
(k) said insect is Psylliodes chrysocephala and said target gene has a DNA sequence selected from the group consisting of SEQ ID NOs:552 - 859;
(1) said insect is a Phyllotreta species and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:860 -
1410;
(m)said insect is Phyllotreta atra (turnip flea beetle), said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:860 - 1155; (n) said insect is Phyllotreta cruciferae (canola flea beetle), said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:1156 - 1391, 1731- 1972, and 1974;
(o) said insect is Phyllotreta striolata (striped flea beetle), said recombinant RNA
molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:1392 - 1410, 1973, and 1975;
(p) said insect is & Psylliodes species and said silencing element comprises an RNA strand having a sequence selected from the group consisting of SEQ ID NOs:1411
- 1718; or
(q) said insect is Psylliodes chrysocephala, said recombinant RNA molecule comprises dsRNA, and said dsRNA comprises at least one RNA sequence selected from the group consisting of SEQ ID NOs:1411 - 1718.
16. The method of claim 6 or 11, or the insecticidal composition of claim 8, wherein:
(a) said plant is selected from an ornamental plant or a crop plant;
(b) said plant is a plant in the family Brassicaceae;
(c) said plant is & Brassica species selected from the group consisting of B. napus, B. juncea, B. carinata, B. rapa, B. oleracea, B. rupestris, B. septiceps, B. nigra, B. narinosa, B. perviridus, B. tournefortii, and B. fructiculosa;
(d) said plant is a Brassica plant selected from the group consisting canola, rapeseed, turnips, and field mustard or turnip rape;
(e) said plant is selected from the group consisting of Glycine max, Linum
usitatissimum, Zea mays, Carthamus tinctorius, Helianthus annuus, Nicotiana tabacum, Arabidopsis thaliana, Betholettia excelsa, Ricinus communis, Cocos nucifera, Coriandrum sativum, Gossypium spp., Arachis hypogaea, Simmondsia chinensis, Solanum tuberosum, Elaeis guineensis, Olea europaea, Oryza sativa, Cucurbita maxim, Hordeum vulgare, and Triticum aestivum; or
(f) said plant is a potato plant and said insect is Epitrix cucumeris (potato flea beetle).
17. The method of claim 11, wherein:
(a) said method further comprises expression in said plant of at least one pesticidal agent selected from the group consisting of a patatin, a plant lectin, a
phytoecdysteroid, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, a bacterium that produces an insecticidal protein, an entomicidal bacterial species, Lysinibacillus sphaericus (Bacillus sphaericus), Brevibacillus laterosporus (Bacillus
laterosporus), Chromobacterium species, Chromobacterium subtsugae,
Paenibacillus species, Paenibacillus lentimorbus, and Paenibacillus popilliae; or (b) said method further comprises expression in said plant of at least one protein
conferring tolerance to an herbicide.
18. The plant having improved resistance to an insect, provided by the method of claim 11, or fruit, seed, or propagatable parts of said plant.
19. A recombinant DNA construct comprising a heterologous promoter operably linked to DNA encoding an RNA transcript comprising at least 21 contiguous nucleotides of a sequence of about 95% to about 100% identity or complementarity with a sequence selected from the group consisting of SEQ ID NOs:860 - 1718 and 1722 - 1730.
20. The recombinant DNA construct of claim 19, wherein said heterologous promoter is functional for expression of said RNA transcript in a bacterium or in a plant cell.
21. A recombinant vector, plant chromosome or plastid, or transgenic plant cell comprising the recombinant DNA construct of claim 19.
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